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Sample records for cell tight junctions

  1. Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

    PubMed Central

    Alanne, Maria H.; Pummi, Kati; Heape, Anthony M.; Grènman, Reidar; Peltonen, Juha; Peltonen, Sirkku

    2009-01-01

    Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009) PMID:19153196

  2. A proposed route to independent measurements of tight junction conductance at discrete cell junctions

    PubMed Central

    Zhou, Lushan; Zeng, Yuhan; Baker, Lane A; Hou, Jianghui

    2015-01-01

    Direct recording of tight junction permeability is of pivotal importance to many biologic fields. Previous approaches bear an intrinsic disadvantage due to the difficulty of separating tight junction conductance from nearby membrane conductance. Here, we propose the design of Double whole-cell Voltage Clamp - Ion Conductance Microscopy (DVC-ICM) based on previously demonstrated potentiometric scanning of local conductive pathways. As proposed, DVC-ICM utilizes two coordinated whole-cell patch-clamps to neutralize the apical membrane current during potentiometric scanning, which in models described here will profoundly enhance the specificity of tight junction recording. Several potential pitfalls are considered, evaluated and addressed with alternative countermeasures. PMID:26716077

  3. Characterization of Tight Junction Proteins in Cultured Human Urothelial Cells

    PubMed Central

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

    2010-01-01

    Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintainance 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 immuno-fluorescence 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. PMID:18553212

  4. Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

    PubMed Central

    PATIBANDLA, PHANI K.; TYAGI, NEETU; DEAN, WILLIAM L.; TYAGI, SURESH C.; ROBERTS, ANDREW M.; LOMINADZE, DAVID

    2009-01-01

    We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. PMID:19507189

  5. Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells

    PubMed Central

    Kaiser, M.; Pereira, S.; Pohl, L.; Ketelhut, S.; Kemper, B.; Gorzelanny, C.; Galla, H. -J.; Moerschbacher, B. M.; Goycoolea, F. M.

    2015-01-01

    Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability. PMID:25970096

  6. Tight junction proteins: from barrier to tumorigenesis.

    PubMed

    Runkle, E Aaron; Mu, David

    2013-08-28

    The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.

  7. Tight Junction Proteins: From Barrier to Tumorigenesis

    PubMed Central

    Runkle, E. Aaron; Mu, David

    2013-01-01

    The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function - that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis. PMID:23743355

  8. Diesel exhaust particles modulate the tight junction protein occludin in lung cells in vitro

    PubMed Central

    Lehmann, Andrea D; Blank, Fabian; Baum, Oliver; Gehr, Peter; Rothen-Rutishauser, Barbara M

    2009-01-01

    Background Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 μg/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. Results Only the highest dose of DEP (125 μg/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. Conclusion We concluded that high concentrations of DEP (125 μg/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells. PMID:19814802

  9. 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.

  10. Analysis of Tight Junction Formation and Integrity

    SciTech Connect

    Karakaya, Mahmut; Kerekes, Ryan A; Morrell-Falvey, Jennifer L; Foster, Carmen M; Retterer, Scott T

    2012-01-01

    In this paper, we study segmentation of tight junctions and analyze the formation and integrity of tight junctions in large-scale confocal image stacks, a challenging biological problem because of the low spatial resolution images and the presence of breaks in tight junction structure. We present an automated, three-step processing approach for tight junction analysis. In our approach, we first localize each individual nucleus in the image by using thresholding, morphological filters and active contours. By using each nucleus position as a seed point, we automatically segment the cell body based on the active contour. We then use an intensity-based skeletonization algorithm to generate the boundary regions for each cell, and features are extracted from tight junctions associated with each cell to assess tight junction continuity. Based on qualitative results and quantitative comparisons, we show that we are able to automatically segment tight junctions and compute relevant features that provide a quantitative measure of tight junction formation to which the permeability of the cell monolayer can ultimately be correlated.

  11. 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

  12. Remodeling of the tight junction during recovery from exposure to hydrogen peroxide in kidney epithelial cells

    PubMed Central

    Gonzalez, Jeannette E.; DiGeronimo, Robert J.; Arthur, D’Ann E.; King, Jonathan M.

    2009-01-01

    Renal ischemia-reperfusion injury results in oxidative stress-induced alterations in barrier function. Activation of the mitogen-activated protein (MAP) kinase pathway during recovery from oxidative stress may be an effector of oxidant-induced tight junction reorganization. We hypothesized that tight junction composition and barrier function would be perturbed during recovery from oxidative stress. We developed a model of short-term H2O2 exposure followed by recovery using Madin Darby Canine Kidney cells (MDCK II). H2O2 perturbs barrier function without a significant cytotoxic effect except in significant doses. ERK-1/2 and p38, both enzymes of the MAP kinase pathway, were activated within minutes of exposure to H2O2. Transient exposure to H2O2 produced a biphasic response in transepithelial electrical resistance (TER). An initial drop in TER at 6 hours was followed by a significant increase at 24 hours. Inhibition of ERK-1/2 activation attenuated the increase in TER observed at 24 hours. Expression of occludin initially decreased followed by partial recovery at 24 hours. In contrast, claudin-1 levels decreased and failed to recover at 24 hours. Claudin-2 levels markedly decreased at 24 hours; however, inhibition of ERK-1/2 activation was protective. Occludin and claudin-1 localization at the apical membrane on immunofluorescent images was fragmented at 6 hours after H2O2 exposure with subsequent recovery of appropriate localization by 24 hours. MDCK II cell recovery after H2O2 exposure is associated with functional and structural modification of the tight junction that are mediated in part by activation of the MAP kinase enzymes, ERK-1/2 and p38. PMID:19733232

  13. Langerhans cells and lymph node dendritic cells express the tight junction component claudin-1.

    PubMed

    Zimmerli, Simone C; Hauser, Conrad

    2007-10-01

    Claudin-1 is a critical structural component of tight junctions that have an important role in adhesive properties, barrier function, and paracellular transport of epithelia and other nonhematopoietic tissues. We found claudin-1 in murine CD207+ Langerhans cells (LC) residing in epidermis. Claudin-1 was not detected in other skin dendritic cells (DC). LC expressed claudin-1 in steady state and inflamed skin. Claudin-1 was demonstrated further in lymph node LC under steady state and inflammatory conditions, including after direct tracking with tetramethylrhodamine-isothiocyanate (TRITC). All subsets of skin draining lymph node DC defined by CD205, CD11b, CD11c, and CD8, including a presumably blood-borne lymph node resident CD8+CD207+ LC population, were claudin-1+. TRITC tracking demonstrated claudin-1 in CD207- skin migrant DC in the lymph node, suggesting upregulation of this molecule during migration or once arrived in the lymph node. Claudin-1 expression in CD207+ cells was confirmed at the protein and mRNA levels. Transforming growth factor-beta, a factor critical for the induction of LC in vitro and in vivo, stimulated the accumulation of claudin-1 mRNA and protein when added to bone marrow cells cultured with GM-CSF and IL-4. Claudin-1 may thus have an important function in adhesion and/or migration of LC.

  14. Nuclear localization of the tight junction protein ZO-2 in epithelial cells.

    PubMed

    Islas, Socorro; Vega, Jesús; Ponce, Lissette; González-Mariscal, Lorenza

    2002-03-10

    The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.

  15. Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells.

    PubMed

    Maia-Brigagão, C; Morgado-Díaz, J A; De Souza, W

    2012-06-01

    Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together constitute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involving the establishment of the infection have not yet been fully elucidated. In this study, we show that after 24h of interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular permeability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance and permeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epithelium viability was not affected. We also demonstrate that there was no change in expression of junctional proteins (tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion was significantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The present work shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbances of the tight, adherens and desmosomal junctions.

  16. Exercise regulation of intestinal tight junction proteins.

    PubMed

    Zuhl, Micah; Schneider, Suzanne; Lanphere, Katherine; Conn, Carole; Dokladny, Karol; Moseley, Pope

    2014-06-01

    Gastrointestinal distress, such as diarrhoea, cramping, vomiting, nausea and gastric pain are common among athletes during training and competition. The mechanisms that cause these symptoms are not fully understood. The stress of heat and oxidative damage during exercise causes disruption to intestinal epithelial cell tight junction proteins resulting in increased permeability to luminal endotoxins. The endotoxin moves into the blood stream leading to a systemic immune response. Tight junction integrity is altered by the phosphoylation state of the proteins occludin and claudins, and may be regulated by the type of exercise performed. Prolonged exercise and high-intensity exercise lead to an increase in key phosphorylation enzymes that ultimately cause tight junction dysfunction, but the mechanisms are different. The purpose of this review is to (1) explain the function and physiology of tight junction regulation, (2) discuss the effects of prolonged and high-intensity exercise on tight junction permeability leading to gastrointestinal distress and (3) review agents that may increase or decrease tight junction integrity during exercise.

  17. Slit Diaphragms Contain Tight Junction Proteins

    PubMed Central

    Fukasawa, Hirotaka; Bornheimer, Scott; Kudlicka, Krystyna; Farquhar, Marilyn G.

    2009-01-01

    Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation. PMID:19478094

  18. Current trends in salivary gland tight junctions

    PubMed Central

    Baker, Olga J.

    2016-01-01

    ABSTRACT Tight junctions form a continuous intercellular barrier between epithelial cells that is required to separate tissue spaces and regulate selective movement of solutes across the epithelium. They are composed of strands containing integral membrane proteins (e.g., claudins, occludin and tricellulin, junctional adhesion molecules and the coxsackie adenovirus receptor). These proteins are anchored to the cytoskeleton via scaffolding proteins such as ZO-1 and ZO-2. In salivary glands, tight junctions are involved in polarized saliva secretion and barrier maintenance between the extracellular environment and the glandular lumen. This review seeks to provide an overview of what is currently known, as well as the major questions and future research directions, regarding tight junction expression, organization and function within salivary glands. PMID:27583188

  19. Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells.

    PubMed

    Li, C X; Poznansky, M J

    1990-12-14

    The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.

  20. Crumbs 3b promotes tight junctions in an ezrin-dependent manner in mammalian cells

    PubMed Central

    Tilston-Lünel, Andrew M.; Haley, Kathryn E.; Schlecht, Nicolas F.; Wang, Yanhua; Chatterton, Abigail L.D.; Moleirinho, Susana; Watson, Ailsa; Hundal, Harinder S.; Prystowsky, Michael B.; Gunn-Moore, Frank J.; Reynolds, Paul A.

    2016-01-01

    Crumbs 3 (CRB3) is a component of epithelial junctions, which has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion, and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, we describe novel data demonstrating that, as with previous studies on CRB3a, CRB3b also promotes the formation of tight junctions (TJs). However, significantly we demonstrate that the 4.1-ezrin–radixin–moesin-binding motif of CRB3b is required for CRB3b functionality and that ezrin binds to the FBM of CRB3b. Furthermore, we show that ezrin contributes to CRB3b functionality and the correct distribution of TJ proteins. We demonstrate that both CRB3 isoforms are required for the production of functionally mature TJs and also the localization of ezrin to the plasma membrane. Finally, we demonstrate that reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and shows evidence that while CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC. PMID:27190314

  1. 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.

  2. Interleukin-34 restores blood-brain barrier integrity by upregulating tight junction proteins in endothelial cells.

    PubMed

    Jin, Shijie; Sonobe, Yoshifumi; Kawanokuchi, Jun; Horiuchi, Hiroshi; Cheng, Yi; Wang, Yue; Mizuno, Tetsuya; Takeuchi, Hideyuki; Suzumura, Akio

    2014-01-01

    Interleukin-34 (IL-34) is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R), and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS) and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood-brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood-brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease.

  3. Interleukin-34 Restores Blood–Brain Barrier Integrity by Upregulating Tight Junction Proteins in Endothelial Cells

    PubMed Central

    Jin, Shijie; Sonobe, Yoshifumi; Kawanokuchi, Jun; Horiuchi, Hiroshi; Cheng, Yi; Wang, Yue; Mizuno, Tetsuya; Takeuchi, Hideyuki; Suzumura, Akio

    2014-01-01

    Interleukin-34 (IL-34) is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R), and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS) and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood–brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood–brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease. PMID:25535736

  4. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

    PubMed

    Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2016-12-13

    Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca(2+)-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

  5. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers

    PubMed Central

    Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S.; Rao, Roshan G.; Shukla, Pradeep K.; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2016-01-01

    Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK. PMID:27958326

  6. HGF and the regulation of tight junctions in human prostate cancer cells.

    PubMed

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

    2014-07-01

    Hepatocyte growth factor (HGF) may impact the metastasis of prostate cancer via its action on prostate stem cells or their progeny. Tight junctions (TJs) are crucial to the process of metastasis and have been previously shown to be regulated by HGF. The present study aimed to evaluate the effect of HGF on the function of TJs in human prostate epithelial, prostate stem cell-like and prostate cancer cell lines. Four human prostate cancer cell lines (PC-3, DU-145, PZHPV-7, CaHPV-10), normal adult prostate parental epithelial cells (RWPE-1) and a stem cell-like derivative of RWPE-1 (WPE-STEM) were used to assess HGF-induced changes in TJs. A significant difference was noted in the behaviour between the WPE-STEM, RWPE-1 and the cancer cell lines which was HGF concentration-dependent. However, in the WPE-STEM cells, the effect was biphasic, with the cells seemingly resistant to HGF-modulated TJ disruption. Closer examination revealed that HGF affected the redistribution of ZO-1, ZO-2 and ZO-3 away from the TJs of confluent cells with concurrent loss of claudin-1 and claudin-5, and western blot analysis revealed a loss in TJ protein expression of ZO-1 and ZO-2. We demonstrated for the first time that HGF regulates TJ function in human prostate cells. Moreover, this regulation was dependent on the tumourigenicity of the cells, with the most aggressive cells most susceptible and the stem cell-like cells least susceptible. These data offer an intriguing glimpse of how TJs affect the behaviour of prostate cancer cells and how HGF modulates the expression and function of the molecules maintaining TJ structure and function.

  7. Claudin-11 and occludin are major contributors to Sertoli cell tight junction function, in vitro

    PubMed Central

    McCabe, Mark J; Foo, Caroline FH; Dinger, Marcel E; Smooker, Peter M; Stanton, Peter G

    2016-01-01

    The Sertoli cell tight junction (TJ) is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% (P < 0.01), 51% (P < 0.01), and 62% (P < 0.01), respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly (P < 0.01) to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant (P < 0.01) contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function. PMID:26585695

  8. Density-enhanced Phosphatase 1 Regulates Phosphorylation of Tight Junction Proteins and Enhances Barrier Function of Epithelial Cells*

    PubMed Central

    Sallee, Jennifer L.; Burridge, Keith

    2009-01-01

    Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. The receptor-tyrosine phosphatase DEP-1 (CD148/PTP-η) has been implicated in cell growth and differentiation as well as in regulating phosphorylation of junctional proteins. However, the role of DEP-1 in regulating tight junction phosphorylation and the integrity of cell-cell junctions is still under investigation. In this study, we used a catalytically dead substrate-trapping mutant of DEP-1 to identify potential substrates at cell-cell junctions. We have shown that in epithelial cells the trapping mutant of DEP-1 interacts with the tight junction proteins occludin and ZO-1 in a tyrosine phosphorylation-dependent manner. In contrast, PTP-PEST, Shp2, and PTPμ did not interact with these proteins, suggesting that the interaction of DEP-1 with occludin and ZO-1 is specific. In addition, occludin and ZO-1 were dephosphorylated by DEP-1 but not these other phosphatases in vitro. Overexpression of DEP-1 increased barrier function as measured by transepithelial electrical resistance and also reduced paracellular flux of fluorescein isothiocyanate-dextran following a calcium switch. Reduced DEP-1 expression by small interfering RNA had a small but significant increase in junction permeability. These data suggest that DEP-1 can modify the phosphorylation state of tight junction proteins and play a role in regulating permeability. PMID:19332538

  9. Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Ye, Dongmei; Ma, Thomas Y; Said, Hamid M

    2007-11-01

    Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

  10. Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico-basal alignment of adherens and tight junctions in epithelial cells.

    PubMed

    Yamada, Tomohiro; Kuramitsu, Kaori; Rikitsu, Etsuko; Kurita, Souichi; Ikeda, Wataru; Takai, Yoshimi

    2013-11-01

    Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial-like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin-based cell-cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)-based cell-cell adhesion, and cadherin and claudin were recruited to the nectin-3- and JAM-based cell-cell adhesion sites to form AJ-like and TJ-like domains, respectively. This inversed alignment of the AJ-like and TJ-like domains was reversed by complementary expression of CPMs Par-3, atypical protein kinase C, Par-6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico-basal alignment of TJs and AJs in epithelial cells.

  11. The effect of nicotine in vitro on the integrity of tight junctions in Caco-2 cell monolayers.

    PubMed

    McGilligan, V E; Wallace, J M W; Heavey, P M; Ridley, D L; Rowland, I R

    2007-09-01

    Ulcerative colitis is characterised by impairment of the epithelial barrier and tight junction alterations resulting in increased intestinal permeability. UC is less common in smokers with smoking reported to decrease paracellular permeability. The aim of this study was thus to determine the effect of nicotine, the major constituent in cigarettes and its metabolites on the integrity of tight junctions in Caco-2 cell monolayers. The integrity of Caco-2 tight junctions was analysed by measuring the transepithelial electrical resistance (TER) and by tracing the flux of the fluorescent marker fluorescein, after treatment with various concentrations of nicotine or nicotine metabolites over 48 h. TER was significantly higher compared to the control for all concentrations of nicotine 0.01-10 microM at 48 h (p<0.001), and for 0.01 microM (p<0.001) and 0.1 microM and 10 microM nicotine (p < 0.01) at 12 and 24 h. The fluorescein flux results supported those of the TER assay. TER readings for all nicotine metabolites tested were also higher at 24 and 48 h only (p < or = 0.01). Western blot analysis demonstrated that nicotine up-regulated the expression of the tight junction proteins occludin and claudin-1 (p < or = 0.01). Overall, it appears that nicotine and its metabolites, at concentrations corresponding to those reported in the blood of smokers, can significantly improve tight junction integrity, and thus, decrease epithelial gut permeability. We have shown that in vitro, nicotine appears more potent than its metabolites in decreasing epithelial gut permeability. We speculate that this enhanced gut barrier may be the result of increased expression of claudin-1 and occludin proteins, which are associated with the formation of tight junctions. These findings may help explain the mechanism of action of nicotine treatment and indeed smoking in reducing epithelial gut permeability.

  12. Epithelial Cell Adhesion Molecule (EpCAM) Regulates Claudin Dynamics and Tight Junctions* ♦

    PubMed Central

    Wu, Chuan-Jin; Mannan, Poonam; Lu, Michael; Udey, Mark C.

    2013-01-01

    Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins. PMID:23486470

  13. Behavior of Primary Cilia and Tricellular Tight Junction Proteins during Differentiation in Temperature-Sensitive Mouse Cochlear Precursor Hair Cells.

    PubMed

    Kakuki, Takuya; Kaneko, Yakuto; Takano, Kenichi; Ninomiya, Takafumi; Kohno, Takayuki; Kojima, Takashi; Himi, Tetsuo

    2016-01-01

    In the sensory hair cells of the mammalian cochlea, the primary cilia in the planar cell polarity as well as the tight junctions in the epithelial cell polarity and the barrier are important to maintain normal hearing. Temperature-sensitive mouse cochlear precursor hair cells were used to investigate the behavior of primary cilia and tricellular tight junction proteins during the differentiation of sensory hair cells. In undifferentiated cells (incubated at 33°C), many acetylated tubulin-positive primary cilia were observed, and each was accompanied with an x03B3;-tubulin-positive basal body. The primary cilia had a '9 + 0' architecture with nine outer microtubule doublets but lacking a central pair of microtubules. In differentiated cells (incubated at 39°C), acetylated tubulin-positive primary cilia as well as acetylated tubulin-positive cilia-like structures were partially observed on the cell surface. In differentiated cells, the number of primary cilia was markedly reduced compared with undifferentiated cells, and innumerable cilia-like structures with no ciliary pockets were partially observed on the cell surface. In undifferentiated cells, few tricellulin molecules and lipolysis-stimulated lipoprotein receptors (LSRs) were observed in the cytoplasm. In differentiated cells, many tricellulin molecules and LSRs were observed on the membranes and within the cytoplasm. Conditional immortalized mouse cochlear precursor hair cells may be useful to investigate the roles of primary cilia and tricellular tight junctions during cellular differentiation and degeneration such as apoptosis.

  14. TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration

    PubMed Central

    Rousseau, Adrien; McEwen, Alastair G.; Poussin-Courmontagne, Pierre; Rognan, Didier; Nominé, Yves; Rio, Marie-Christine; Tomasetto, Catherine; Alpy, Fabien

    2013-01-01

    Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration. PMID:24311986

  15. Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

    PubMed

    Nemoto, Ryo; Yamamoto, Shintaro; Ogawa, Tomohisa; Naude, Ryno; Muramoto, Koji

    2015-05-05

    The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

  16. Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism.

    PubMed

    Dunagan, Mitzi; Chaudhry, Kamaljit; Samak, Geetha; Rao, R K

    2012-12-15

    Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200-600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

  17. Tight junction between endothelial cells: the interaction between nanoparticles and blood vessels

    PubMed Central

    Zhang, Yue

    2016-01-01

    Summary Since nanoparticles are now widely applied as food additives, in cosmetics and other industries, especially in medical therapy and diagnosis, we ask here whether nanoparticles can cause several adverse effects to human health. In this review, based on research on nanotoxicity, we mainly discuss the negative influence of nanoparticles on blood vessels in several aspects and the potential mechanism for nanoparticles to penetrate endothelial layers of blood vessels, which are the sites of phosphorylation of tight junction proteins (claudins, occludins, and ZO (Zonula occludens)) proteins, oxidative stress and shear stress. We propose a connection between the presence of nanoparticles and the regulation of the tight junction, which might be the key approach for nanoparticles to penetrate endothelial layers and then have an impact on other tissues and organs. PMID:27335757

  18. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  19. L. plantarum prevents Enteroinvasive Escherichia coli-induced tight junction proteins changes in intestinal epithelial cells

    PubMed Central

    2009-01-01

    Background It is increasingly recognized that Lactobacillus plantarum (L. plantarum) has the ability to protect against Enteropathogenic Escherichia coli (EPEC)-induced damage of the epithelial monolayer barrier function by preventing changes in host cell morphology, attaching/effacing (A/E) lesion formation, monolayer resistance, and macromolecular permeability. However, the cellular mechanism involved in this protective effect still remained to be clarified. Methods This study was to investigate the effect of L. plantarum on the changes of Caco-2 cells responding to Enteroinvasive Escherichia coli (EIEC), the permeability of cell monolayer and the transmissivity of dextran, and the distribution and expression of the tight junction (TJ) proteins, such as Claudin-1, Occludin, JAM-1 and ZO-1 were examined when infected with EIEC or adhesived of L. plantarum after infection by confocal laser scanning microscopy (CLSM), immunohistochemistry and Western blotting, the cytoskeleton protein F-actin were observed with FITC-phalloidin. Results This study demonstrated that the transepithelial electrical resistance (TER) step down and dextran integrated intensity (DII) step up with time after infected with EIEC, but after treating with L. plantarum, the changes of TER and DII were improved as compared with EIEC group. L. plantarum prevented the damage of expression and rearrangement of Claudin-1, Occludin, JAM-1 and ZO-1 proteins induced by EIEC, and could ameliorate the injury of cytoskeleton protein F-actin infected with EIEC. Conclusion L. plantarum exerted a protective effect against the damage to integrity of Caco-2 monolayer cells and the structure and distribution of TJ proteins by EIEC infection. PMID:19331693

  20. Tight junction proteins expression and modulation in immune cells and multiple sclerosis

    PubMed Central

    Mandel, Ilana; Paperna, Tamar; Glass-Marmor, Lea; Volkowich, Anat; Badarny, Samih; Schwartz, Ilya; Vardi, Pnina; Koren, Ilana; Miller, Ariel

    2012-01-01

    Abstract The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre-treatment levels and clinical response phenotype to interferon-β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune-mediated diseases such as MS. PMID:21762372

  1. 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.

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

    SciTech Connect

    Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret; Kusch, Angelika; Korenbaum, Elena; Haller, Hermann; Dumler, Inna

    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), 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.

  3. HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

    PubMed

    Sufiawati, Irna; Tugizov, Sharof M

    2014-01-01

    Herpes simplex virus (HSV) types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD). Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

  4. Effect of caveolin-1 on the expression of tight junction-associated proteins in rat glioma-derived microvascular endothelial cells

    PubMed Central

    Li, Yao; Liu, Li-Bo; Ma, Teng; Wang, Ping; Xue, Yi-Xue

    2015-01-01

    Caveolin-1 affects the permeability of blood-tumor barrier (BTB) by regulating the expression of tight junction-associated proteins. However, the effect is still controversial. In the present work, we studied the regulative effect of caveolin-1 on the expression of tight junction-associated proteins and BTB via directly silencing and overexpressing of caveolin-1 by recombinant adenovirus transduction of glioma-derived microvascular endothelial cells in rat brain. The results show that the caveolin-1 downregulation resulted in decreased expression of tight junction-associated proteins, opening of tight junctions, and increasing the permeability of BTB, whereas the overexpression of caveolin-1 presented the opposite effects. Therefore, we conclude that caveolin-1 regulates the expression of tight junction-associated proteins in a positive manner, which further plays a role in the regulation of BTB permeability. This finding provides a novel therapeutic target for selectively opening of BTB. PMID:26722502

  5. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

    PubMed

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; Del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C; Hayes, Peter C; Plevris, John N; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J

    2017-01-30

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

  6. Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

    PubMed Central

    Gamal, Wesam; Treskes, Philipp; Samuel, Kay; Sullivan, Gareth J.; Siller, Richard; Srsen, Vlastimil; Morgan, Katie; Bryans, Anna; Kozlowska, Ada; Koulovasilopoulos, Andreas; Underwood, Ian; Smith, Stewart; del-Pozo, Jorge; Moss, Sharon; Thompson, Alexandra Inés; Henderson, Neil C.; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre-Olivier; Nelson, Leonard J.

    2017-01-01

    Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization. PMID:28134251

  7. aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation.

    PubMed

    Iden, Sandra; Misselwitz, Steve; Peddibhotla, Swetha S D; Tuncay, Hüseyin; Rehder, Daniela; Gerke, Volker; Robenek, Horst; Suzuki, Atsushi; Ebnet, Klaus

    2012-03-05

    The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.

  8. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers.

    PubMed

    Peng, Luying; Li, Zhong-Rong; Green, Robert S; Holzman, Ian R; Lin, Jing

    2009-09-01

    Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

  9. Regulation and roles for claudin-family tight junction proteins

    PubMed Central

    Findley, Mary K.; Koval, Michael

    2009-01-01

    Transmembrane proteins known as claudins play a critical role in tight junctions by regulating paracellular barrier permeability. The control of claudin assembly into tight junctions requires a complex interplay between several classes of claudins, other transmembrane proteins and scaffold proteins. Claudins are also subject to regulation by post-translational modifications including phosphorylation and palmitoylation. Several human diseases have been linked to claudin mutations, underscoring the physiologic function of these proteins. Roles for claudins in regulating cell phenotype and growth control also are beginning to emerge, suggesting a multifaceted role for claudins in regulation of cells beyond serving as a simple structural element of tight junctions. PMID:19319969

  10. Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

    PubMed

    Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

    2017-11-01

    Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

  11. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.

    PubMed

    Torres-Flores, Jesús M; Silva-Ayala, Daniela; Espinoza, Marco A; López, Susana; Arias, Carlos F

    2015-01-15

    Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

  12. Tricellular Tight Junctions in the Inner Ear

    PubMed Central

    2016-01-01

    Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs. PMID:27195292

  13. Cortisol affects tight junction morphology between pavement cells of rainbow trout gills in single-seeded insert culture.

    PubMed

    Sandbichler, Adolf Michael; Farkas, Julia; Salvenmoser, Willi; Pelster, Bernd

    2011-12-01

    A primary culture system of rainbow trout gill pavement cells grown on permeable support (single-seeded insert, SSI) was used to examine histological and physiological changes induced by the addition of the corticosteroid hormone cortisol. Pavement cell epithelia were cultured under symmetrical conditions (L15 apical/L15 basolateral) and developed a high transepithelial resistance (TER, 6.84 ± 1.99 kΩ cm(2), mean ± SEM) with a low phenol red diffusion rate (PRD, 0.15 ± 0.03 μmol l(-1)/day). Addition of cortisol to the basolateral compartment increased TER twofold and reduced PRD threefold over a 5-day period. A similar increase in TER could be seen after 24 h apical freshwater (FW) in control cultures. In cortisol-treated cultures FW exposure did not change TER, but PRD increased significantly. Histochemical staining of the cytoskeleton of cells in SSI culture revealed a morphological partitioning into a single mucosal layer of polarized, polygonal cells featuring cortical F-actin rings which were comparable to F-actin rings of epithelial cells on the lamellar and filamental surface, and several unorganized serosal layers of cells with F-actin stress fibers. Addition of cortisol increased cell density by 18% and in the mucosal layer it led to smaller, less polygonal cells with increased height and increased cell contact area. In transmission electron microscopic images two pairs of cytoplasmatic electron-dense structures confining the zonula occludens apically and basally toward the zonula adhaerens were found. Addition of cortisol increased the distance between those paired structures, hence led to deeper tight junctions. The cortisol-induced increase in barrier properties, therefore, involves a structural fortification of the tight junctions which was not generally modified by a short 24-h apical freshwater stress. These results identify cortisol as a regulator of tight junction morphology between pavement cells of euryhaline fish such as the

  14. JAM-A and aPKC: A close pair during cell-cell contact maturation and tight junction formation in epithelial cells.

    PubMed

    Ebnet, Klaus

    2013-01-01

    Cell-cell adhesion plays a critical role in the formation of barrier-forming epithelia. The molecules which mediate cell-cell adhesion frequently act as signaling molecules by recruiting and/or assembling cytoplasmic protein complexes. Junctional Adhesion Molecule (JAM)-A interacts with the cell polarity protein PAR-3, a member of the PAR-3-aPKC-PAR-6 complex, which regulates the formation of cell-cell contacts and the development of tight junctions (TJs). In our recent study we found that JAM-A is localized at primordial, spot-like cell-cell junctions (pAJs) in a non-phosphorylated form. After the recruitment of the PAR-aPKC complex and its activation at pAJs, aPKC phosphorylates JAM-A at Ser285 to promote the maturation of immature junctions. In polarized epithelial cells, aPKC phosphorylates JAM-A selectively at the TJs to maintain the barrier function of TJs. Thus, through mutual regulation, JAM-A and aPKC form a functional unit that regulates the establishment of barrier-forming junctions in vertebrate epithelial cells.

  15. Involvement of the helix-loop-helix protein Id-1 in the glucocorticoid regulation of tight junctions in mammary epithelial cells.

    PubMed

    Woo, P L; Cercek, A; Desprez, P Y; Firestone, G L

    2000-09-15

    Mammary epithelial cell-cell junctions undergo morphological and structural differentiation during pregnancy and lactation, but little is known about the transcriptional regulators that are involved in this process. In Con8 mammary epithelial tumor cells, we have previously documented that the synthetic glucocorticoid, dexamethasone, induces the reorganization of the tight junction and adherens junction and stimulates the monolayer transepithelial electrical resistance (TER), a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment rapidly and strongly stimulated the level of the Id-1 protein, which is a serum-inducible helix-loop-helix transcriptional repressor. The steroid induction of Id-1 was robust by 4 h of treatment and maintained over a 24-h period. Isopropyl-1-thio-beta-d-galactopyranoside-inducible expression of exogenous Id-1 in Con8 cells was shown to strongly facilitate the dexamethasone induction of TER in the absence of serum without altering the dexamethasone-dependent reorganization of ZO-1, beta-catenin, or F-actin. Ectopic overexpression of Id-1 in the SCp2 nontumorigenic mammary epithelial cells, which does not undergo complete dexamethasone-dependent tight junction reorganization, enhanced the dexamethasone-induced ZO-1 tight junction localization and stimulated the monolayer TER. Moreover, antisense reduction of Id-1 protein in SCp2 cells prevented the apical junction reorganization and dexamethasone-stimulated TER. Our results implicate Id-1 as acting as a critical regulator of mammary epithelial cell-cell interactions at an early step in the glucocorticoid-dependent signaling pathway that controls tight junction integrity.

  16. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

    SciTech Connect

    McCall, Ingrid C.; Betanzos, Abigail; Weber, Dominique A.; Nava, Porfirio; Miller, Gary W.; Parkos, Charles A.

    2009-11-15

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

  17. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

    PubMed Central

    McCall, Ingrid C.; Betanzos, Abigail; Weber, Dominique A.; Nava, Porfirio; Miller, Gary W.; Parkos, Charles A.

    2010-01-01

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains. PMID:19679145

  18. Dexamethasone suppresses JMJD3 gene activation via a putative negative glucocorticoid response element and maintains integrity of tight junctions in brain microvascular endothelial cells.

    PubMed

    Na, Wonho; Shin, Jee Y; Lee, Jee Y; Jeong, Sangyun; Kim, Won-Sun; Yune, Tae Y; Ju, Bong-Gun

    2017-01-01

    The blood-brain barrier (BBB) exhibits a highly selective permeability to support the homeostasis of the central nervous system (CNS). The tight junctions in the BBB microvascular endothelial cells seal the paracellular space to prevent diffusion. Thus, disruption of tight junctions results in harmful effects in CNS diseases and injuries. It has recently been demonstrated that glucocorticoids have beneficial effects on maintaining tight junctions in both in vitro cell and in vivo animal models. In the present study, we found that dexamethasone suppresses the expression of JMJD3, a histone H3K27 demethylase, via the recruitment of glucocorticoid receptor α (GRα) and nuclear receptor co-repressor (N-CoR) to the negative glucocorticoid response element (nGRE) in the upstream region of JMJD3 gene in brain microvascular endothelial cells subjected to TNFα treatment. The decreased JMJD3 gene expression resulted in the suppression of MMP-2, MMP-3, and MMP-9 gene activation. Dexamethasone also activated the expression of the claudin 5 and occludin genes. Collectively, dexamethasone attenuated the disruption of the tight junctions in the brain microvascular endothelial cells subjected to TNFα treatment. Therefore, glucocorticoids may help to preserve the integrity of the tight junctions in the BBB via transcriptional and post-translational regulation following CNS diseases and injuries.

  19. Inhibition of matrix metalloproteinase activities and tightening of tight junctions by diallyl disulfide in AGS human gastric carcinoma cells.

    PubMed

    Park, Hyun Soo; Kim, Gi-Young; Choi, Il-Whan; Kim, Nam Deuk; Hwang, Hye Jin; Choi, Young-Whan; Choi, Yung Hyun

    2011-05-01

    The effect of diallyl disulfide (DADS), a major component of an oil-soluble allyl sulfide garlic (Allium sativum) derivative, on the correlation between anti-invasive activity and tightening of tight junctions (TJs) was investigated in human gastric adenocarcinoma AGS cells. Our data indicated that the inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance. Activities of matrix metalloprotease (MMP)-2 and -9 in AGS cells were dose-dependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins; however, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 mRNA levels and proteins were increased. Additionally, immunoblotting results indicated that DADS repressed the levels of claudin proteins (claudin-2, -3, and -4), major components of TJs that play key roles in control and selectivity of paracellular transport. Although further studies are needed, these results suggest that DADS treatment may inhibit tumor cell motility and invasion and, therefore, act as a dietary source to decrease the risk of cancer metastasis.

  20. Lecithin-Bound Iodine Prevents Disruption of Tight Junctions of Retinal Pigment Epithelial Cells under Hypoxic Stress

    PubMed Central

    Sugimoto, Masahiko; Kondo, Mineo

    2016-01-01

    Aim. We investigated whether lecithin-bound iodine (LBI) can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia. Method. Cultured human retinal pigment epithelial (ARPE-19) cells were pretreated with LBI. To mimic hypoxic conditions, cells were incubated with CoCl2. We compared the integrity of the tight junctions (TJs) of control to cells with either LBI alone, CoCl2 alone, or LBI + CoCl2. The levels of cytokines in the conditioned media were also determined. Results. Significant decrease in the zonula occludens-1 (ZO-1) intensity in the CoCl2 group compared to the control (5787.7 ± 4126.4 in CoCl2 group versus 29244.6 ± 2981.2 in control; average ± standard deviation). But the decrease was not significant in the LBI + CoCl2 (27189.0 ± 11231.1). The levels of monocyte chemoattractant protein-1 (MCP-1) and Chemokine (C-C Motif) Ligand 11 (CCL-11) were significantly higher in the CoCl2 than in the control (340.8 ± 43.3 versus 279.7 ± 68.3 pg/mL for MCP-1, and 15.2 ± 12.9 versus 12.5 ± 6.1 pg/mL for CCL-11. With LBI pretreatment, the levels of both cytokines were decreased to 182.6 ± 23.8 (MCP-1) and 5.46 ± 1.9 pg/mL for CCL-11). Blockade of MCP-1 or CCL-11 also shows similar result representing TJ protection from hypoxic stress. Conclusions. LBI results in a protective action from hypoxia. PMID:27340563

  1. Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

    PubMed Central

    Yamaguchi, Hiroshi; Kojima, Takashi; Ito, Tatsuya; Kimura, Yasutoshi; Imamura, Masafumi; Son, Seiichi; Koizumi, Jun-ichi; Murata, Masaki; Nagayama, Minoru; Nobuoka, Takayuki; Tanaka, Satoshi; Hirata, Koichi; Sawada, Norimasa

    2010-01-01

    In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-α inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-δ inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control. PMID:20566751

  2. Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

    PubMed Central

    Wiechmann, Allan F.; Ceresa, Brian P.; Howard, Eric W.

    2014-01-01

    Background and Objectives The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium. Methodology/Principal Findings Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime. Conclusions/Significance MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and

  3. 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.

  4. Effects of Soybean Agglutinin on Mechanical Barrier Function and Tight Junction Protein Expression in Intestinal Epithelial Cells from Piglets

    PubMed Central

    Pan, Li; Qin, Guixin; Zhao, Yuan; Wang, Jun; Liu, Feifei; Che, Dongsheng

    2013-01-01

    In this study, we sought to investigate the role of soybean agglutinin (SBA) in mediating membrane permeability and the mechanical barrier function of intestinal epithelial cells. The IPEC-J2 cells were cultured and treated with 0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg/mL SBA. Transepithelial electrical resistance (TEER) and alkaline phosphatase (AP) activity were measured to evaluate membrane permeability. The results showed a significant decrease in TEER values (p < 0.05) in a time- and dose-dependent manner, and a pronounced increase in AP activity (p < 0.05). Cell growth and cell morphology were used to evaluate the cell viability. A significant cell growth inhibition (p < 0.05) and alteration of morphology were observed when the concentration of SBA was increased. The results of western blotting showed that the expression levels of occludin and claudin-3 were decreased by 31% and 64% compared to those of the control, respectively (p < 0.05). In addition, immunofluorescence labeling indicated an obvious decrease in staining of these targets and changes in their localizations. In conclusion, SBA increased the membrane permeability, inhibited the cell viability and reduced the levels of tight junction proteins (occludin and claudin-3), leading to a decrease in mechanical barrier function in intestinal epithelial cells. PMID:24189218

  5. 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

  6. Activation of classical estrogen receptor subtypes reduces tight junction disruption of brain endothelial cells under ischemia/reperfusion injury.

    PubMed

    Shin, Jin A; Yoon, Joo Chun; Kim, Minsuk; Park, Eun-Mi

    2016-03-01

    Ischemic stroke, which induces oxidative stress in the brain, disrupts tight junctions (TJs) between brain endothelial cells, resulting in blood-brain barrier (BBB) breakdown and brain edema. Estrogen reduces oxidative stress and protects brain endothelial cells from ischemic insult. The aim of this study was to determine the protective effects of estrogen on TJ disruption and to examine the roles of classical estrogen receptor (ER) subtypes, ERα- and ERβ, in estrogen effects in brain endothelial cells (bEnd.3) exposed to oxygen-glucose deprivation/reperfusion (OGD/R) injury. Estrogen pretreatment prevented OGD/R-induced decreases in cell viability and TJ protein levels. ERα- and ERβ-specific agonists also reduced TJ disruption. Knockdown of ERα or ERβ expression partially inhibited the effects of estrogen, but completely reversed the effects of corresponding ER subtype-specific agonists on the outcomes of OGD/R. During the early reperfusion period, activation of extracellular signal-regulated kinase1/2 and hypoxia-inducible factor 1α/vascular endothelial growth factor was associated with decreased expression of occludin and claudin-5, respectively, and these changes in TJ protein levels were differentially regulated by ER subtype-specific agonists. Our results suggest that ERα and ERβ activation reduce TJ disruption via inhibition of signaling molecules after ischemic injury and that targeting each ER subtype can be a useful strategy for protecting the BBB from ischemic stroke in postmenopausal women.

  7. The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells

    PubMed Central

    Shimada, Hiroshi; Satohisa, Seiro; Kohno, Takayuki; Takahashi, Syunta; Hatakeyama, Tsubasa; Konno, Takumi; Tsujiwaki, Mitsuhiro; Saito, Tsuyoshi; Kojima, Takashi

    2016-01-01

    Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer. PMID:27036040

  8. The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells.

    PubMed

    Shimada, Hiroshi; Satohisa, Seiro; Kohno, Takayuki; Takahashi, Syunta; Hatakeyama, Tsubasa; Konno, Takumi; Tsujiwaki, Mitsuhiro; Saito, Tsuyoshi; Kojima, Takashi

    2016-05-10

    Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer.

  9. Testosterone Regulates Tight Junction Proteins and Influences Prostatic Autoimmune Responses

    PubMed Central

    Meng, Jing; Mostaghel, Elahe A.; Vakar-Lopez, Funda; Montgomery, Bruce; True, Larry; Nelson, Peter S.

    2015-01-01

    Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology. PMID:21761342

  10. Somatostatin regulates tight junction proteins expression in colitis mice.

    PubMed

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P<0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P<0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P<0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P<0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.

  11. Somatostatin regulates tight junction proteins expression in colitis mice

    PubMed Central

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression. PMID:24966923

  12. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

    PubMed

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A; Wertheimer, Joshua; Mullin, James M

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

  13. Physiology and Function of the Tight Junction

    PubMed Central

    Anderson, James M.; Van Itallie, Christina M.

    2009-01-01

    Understanding of tight junctions has evolved from their historical perception as inert solute barriers to recognition of their physiological and biochemical complexity. Many proteins are specifically localized to tight junctions, including cytoplasmic actin-binding proteins and adhesive transmembrane proteins. Among the latter are claudins, which are critical barrier proteins. Current information suggests that the paracellular barrier is most usefully modeled as having two physiologic components: a system of charge-selective small pores, 4 Å in radius, and a second pathway created by larger discontinuities in the barrier, lacking charge or size discrimination. The first pathway is influenced by claudin expression patterns and the second is likely controlled by different proteins and signals. Recent information on claudin function and disease-causing mutations have led to a more complete understanding of their role in barrier formation, but progress is impeded by lack of high resolution structural information. PMID:20066090

  14. Imaging analysis of the gliadin direct effect on tight junctions in an in vitro three-dimensional Lovo cell line culture system.

    PubMed

    Elli, Luca; Roncoroni, Leda; Doneda, Luisa; Ciulla, Michele M; Colombo, Roberto; Braidotti, Paola; Bonura, Antonella; Bardella, Maria Teresa

    2011-02-01

    Tight junctions play a pivotal role in maintaining the integrity of the intestinal barrier. Their alteration is involved in the pathogenesis of celiac disease. Our aim was to investigate the gliadin effect on the tight junction proteins in an in vitro three-dimensional cell culture model through imaging analyses. Lovo multicellular spheroids were treated with enzymatically digested (PT) gliadin 500 μg/mL and its effect on actin, occludin and zonula occludens-1, was evaluated by means of confocal laser microscopy, transmission electron microscopy and image capture analysis. Compared to untreated spheroids, PT-gliadin-treated ones showed enlargement of the paracellular spaces (9.0±6.9 vs. 6.2±1.7 nm, p<0.05) at transmission electron microscopy and tight junction protein alterations at confocal microscopy and image analyses. In untreated cell cultures thickness of the fluorescence contour of actin, zonula occludens-1 and occludin appeared significantly larger and more intense than in the treated ones. In occludin planimetric analysis the lengths of the integral uninterrupted cellular contour appeared longer in untreated than in PT-gliadin treated spheroids (71.8±42.8 vs. 23.4±25.9 μm, p<0.01). Our data demonstrated that tight junction proteins are directly damaged by gliadin as shown by means of quantitative imaging analysis.

  15. The effect of phytic acid on tight junctions in the human intestinal Caco-2 cell line and its mechanism.

    PubMed

    Fu, Qingxue; Wang, Huizhen; Xia, Mengxin; Deng, Bing; Shen, Hongyi; Ji, Guang; Li, Guowen; Xie, Yan

    2015-12-01

    This study investigated the effect of phytic acid (IP6), a potential absorption enhancer of flavonoid components, on tight junction (TJ) integrity in Caco-2 cell monolayers and its possible mechanisms. Transepithelial electrical resistance (TEER) across the monolayers decreased rapidly, and the flux of fluorescein sodium (a paracellular marker) increased after treating with IP6 in a concentration-dependent manner. Confocal microscopy results showed that IP6 produced a concentration-dependent attenuation in the distribution of occludin, ZO-1, and claudin-1. Immunoblot analysis revealed that IP6 could down-regulate the expression level of these TJ proteins, which resulted in the opening of TJ. Additionally, the divalent cations Ca(2+) and Mg(2+) influenced the IP6-induced distribution of occludin, ZO-1, and claudin-1 in different directions, which enhanced barrier function. In conclusion, IP6 can decrease the integrity of Caco-2 cell monolayers by modulating the TJ proteins' localization and down-regulating the expression levels of TJ proteins including claudin-1, occludin, and ZO-1; the reduction effects of divalent cations such as Ca(2+) and Mg(2+) on the regulation of TJ induced by IP6 should be addressed. The present work will offer some useful guidance for the application of IP6 in drug delivery area.

  16. Collagen peptides ameliorate intestinal epithelial barrier dysfunction in immunostimulatory Caco-2 cell monolayers via enhancing tight junctions.

    PubMed

    Chen, Qianru; Chen, Oliver; Martins, Isabela M; Hou, Hu; Zhao, Xue; Blumberg, Jeffrey B; Li, Bafang

    2017-03-22

    Dysfunction of the intestinal barrier plays a key role in the pathogenesis of inflammatory bowel disease (IBD) and multiple organ failure. The effect of Alaska pollock skin-derived collagen and its 3 tryptic hydrolytic fractions, HCP (6 kDa retentate), MCP (3 kDa retentate) and LCP (3 kDa permeate) on TNF-α induced barrier dysfunction was investigated in Caco-2 cell monolayers. TNF-α induced barrier dysfunction was significantly attenuated by the collagen and its peptide fractions, especially LCP, compared to TNF-α treated controls (P < 0.05). Compared to a negative control, 24 h pre-incubation with 2 mg mL(-1) LCP significantly alleviated the TNF-α induced breakdown of the tight junction protein ZO-1 and occludin and inhibited MLC phosphorylation and MLCK expression. The activation of NFκB and Elk-1 was suppressed by LCP. Thus, collagen peptides may attenuate TNF-α induced barrier dysfunction of Caco-2 cells by inhibiting the NFκB and ERK1/2-mediated MLCK pathway with associated decreases in ZO-1 and occludin protein expression.

  17. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  18. Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells.

    PubMed

    Bulldan, Ahmed; Dietze, Raimund; Shihan, Mazen; Scheiner-Bobis, Georgios

    2016-08-01

    In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy. In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn(2+)transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells. The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility.

  19. Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    PubMed Central

    Zhang, Cong; Yan, Junkai; Xiao, Yongtao; Shen, Yujie; Wang, Jiazheng; Ge, Wensong; Chen, Yingwei

    2017-01-01

    Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers. PMID:28106723

  20. The PIKfyve Inhibitor YM201636 Blocks the Continuous Recycling of the Tight Junction Proteins Claudin-1 and Claudin-2 in MDCK cells

    PubMed Central

    Dukes, Joseph D.

    2012-01-01

    Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation. PMID:22396724

  1. Matrix Metalloproteinase-2 and -9 Secreted by Leukemic Cells Increase the Permeability of Blood-Brain Barrier by Disrupting Tight Junction Proteins

    PubMed Central

    Feng, Saran; Cen, Jiannong; Huang, Yihong; Shen, Hongjie; Yao, Li; Wang, Yuanyuan; Chen, Zixing

    2011-01-01

    Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia. PMID:21857898

  2. Tight Junction Pore and Leak Pathways: A Dynamic Duo

    PubMed Central

    Raleigh, David R.; Yu, Dan; Turner, Jerrold R.

    2015-01-01

    Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function. PMID:20936941

  3. Use of Z310 Cells as an In Vitro Blood-Cerebrospinal Fluid Barrier Model: Tight Junction Proteins and Transport Properties

    PubMed Central

    Shi, Lewis Zhichang; Li, G. Jane; Wang, Shunzhen; Zheng, Wei

    2009-01-01

    Immortalized rat choroidal epithelial Z310 cells have the potential to become an in vitro model for studying transport of materials at blood-cerebrospinal fluid barrier (BCB) (Shi and Zheng, Brain Research 1057:37-48, 2005). This study was designed to demonstrate the presence of tight junction properties in Z310 cells and the functionality of Z310 monolayer in transport of selected model compounds. Western blot analyses revealed the presence of claudin-1, ZO-1, and occludin in Z310 cells. Transmission electron microscopy showed a “tight junction” type of structure in the sub-apical lateral membranes between adjacent Z310 cells. Real-time RT-PCR revealed that Z310 cells expressed representative transporters such as DMT1, MTP1, TfR, p-glycoprotein, ATP7A, ZnT1, ABCC1, Oat3, OCT1 and OB-Ra. Moreover, Z310 cells cultured in a two-chamber Transwell device possessed the ability to transport zidovudine (anionic drug), thyroxine (hormone), thymidine (nucleoside), and leptin (large polypeptide) with kinetic properties similar to those obtained from the in vitro model based on primary culture of choroidal epithelial cells. Taken together, these data indicate that the Z310 BCB model expresses major tight junction proteins and forms a tight barrier in vitro. The model also exhibits the ability to transport substances of various categories across the barrier. PMID:17825520

  4. Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis: effect of interleukin 6 on Sertoli cell tight junctions.

    PubMed

    Pérez, Cecilia Valeria; Sobarzo, Cristian Marcelo; Jacobo, Patricia Verónica; Pellizzari, Eliana Herminia; Cigorraga, Selva Beatriz; Denduchis, Berta; Lustig, Livia

    2012-11-01

    Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.

  5. 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.

  6. Endocytosis and Recycling of Tight Junction Proteins in Inflammation

    PubMed Central

    Utech, Markus; Mennigen, Rudolf; Bruewer, Matthias

    2010-01-01

    A critical function of the epithelial lining is to form a barrier that separates luminal contents from the underlying interstitium. This barrier function is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised under inflammatory conditions. In intestinal epithelial cells, proinflammatory cytokines, for example, interferon-gamma (IFN-γ), induce internalization of TJ proteins by endocytosis. Endocytosed TJ proteins are passed into early and recycling endosomes, suggesting the involvement of recycling of internalized TJ proteins. This review summarizes mechanisms by which TJ proteins under inflammatory conditions are internalized in intestinal epithelial cells and point out comparable mechanism in nonintestinal epithelial cells. PMID:20011071

  7. Tight junctions, tight junction proteins and paracellular permeability across the gill epithelium of fishes: a review.

    PubMed

    Chasiotis, Helen; Kolosov, Dennis; Bui, Phuong; Kelly, Scott P

    2012-12-01

    Paracellular permeability characteristics of the fish gill epithelium are broadly accepted to play a key role in piscine salt and water balance. This is typically associated with differences between gill epithelia of teleost fishes residing in seawater versus those in freshwater. In the former, the gill is 'leaky' to facilitate Na(+) secretion and in the latter, the gill is 'tight' to limit passive ion loss. However, studies in freshwater fishes also suggest that varying epithelial 'tightness' can impact ionoregulatory homeostasis. Paracellular permeability of vertebrate epithelia is largely controlled by the tight junction (TJ) complex, and the fish gill is no exception. In turn, the TJ complex is composed of TJ proteins, the abundance and properties of which determine the magnitude of paracellular solute movement. This review provides consolidated information on TJs in fish gills and summarizes recent progress in research that seeks to understand the molecular composition of fish gill TJ complexes and what environmental and systemic factors influence those components.

  8. 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

  9. Developmental Changes in Proximal Tubule Tight Junction Proteins

    PubMed Central

    HADDAD, MAHA; LIN, FANGMING; DWARAKANATH, VANGIPURAM; CORDES, KIMBERLY; BAUM, MICHEL

    2014-01-01

    We demonstrated previously that neonatal proximal tubules have a lower passive paracellular permeability to chloride ions and higher resistance than that of adult proximal tubules. In addition, administration of thyroid hormone to neonates, before the normal maturational increase in serum thyroid hormone levels, prematurely accelerates the developmental increase in chloride permeability to adult levels. To test the hypothesis that there is a maturational change in tight junction proteins and that thyroid hormone mediates these changes, we examined the two known tight junction proteins present in proximal tubules, occludin and claudin 2. Using immunoblot and immunohistochemistry, we demonstrated that claudin 2 has a 4-fold greater abundance in neonatal proximal tubules than in adult tubules. Occludin, however, has a 4-fold greater expression in adult tubules than in neonatal tubules. Administration of thyroid hormone to neonates did not affect claudin 2 expression, occludin expression, or the transepithelial resistance in rat proximal tubule cells in vitro. In conclusion, there are postnatal maturational changes in tight junction proteins. The factors that cause these maturational changes are unknown but unlikely to be due solely to the maturational increase in thyroid hormone. PMID:15585672

  10. Depletion of Caco-2 cell cholesterol disrupts barrier function by altering the detergent solubility and distribution of specific tight-junction proteins

    PubMed Central

    2004-01-01

    In the present study, we have investigated the role of cholesterol in maintaining the barrier properties of the model intestinal cell line Caco-2. We have extracted membrane cholesterol using methyl-β-cyclodextrin and demonstrated that maximally, methyl-β-cyclodextrin lowered cell cholesterol levels by 40–45%. Depletion of cell cholesterol was accompanied by an 80–90% decrease in monolayer transepithelial electrical resistance and a significant increase in the paracellular permeability of dextrans of 4, 10 and 40 kDa. The increase in dextran permeability was most pronounced for the two lower molecular mass species. In addition to the decline in the barrier properties of the monolayers, extraction of cell cholesterol produced an increase in the Triton X-100 solubility of claudin 3, claudin 4 and occludin, and the loss of all three proteins from the plasma membrane (tight junctions). In contrast, removal of cholesterol had no detectable influence on the detergent solubility or morphological distribution of claudin 1. These results indicate that membrane cholesterol is a critical factor in maintaining the barrier property of epithelial monolayers. More specifically, cholesterol appears to stabilize the association of certain proteins with the tight junctions. PMID:15500448

  11. The Role of IRE-XBP1 Pathway in Regulation of Retinal Pigment Epithelium Tight Junctions

    PubMed Central

    Ma, Jacey H.; Wang, Joshua J.; Li, Junhua; Pfeffer, Bruce A.; Zhong, Yiming; Zhang, Sarah X.

    2016-01-01

    Purpose The retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina. Herein, we investigated the role of X-box binding protein 1 (XBP1), an endoplasmic reticulum (ER) stress-responsive transcription factor, in regulation of RPE tight junctions. Methods Human RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4μ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined. Results Induction of ER stress by thapsigargin, but not tunicamycin, disrupted RPE tight junctions in ARPE-19 cells. Inhibition of XBP1 activation by 4μ8C resulted in a remarkable downregulation of tight junction proteins (ZO-1 and occludin) and defects in tight junction formation in the presence or absence of ER stress inducers. Overexpression of active XBP1 partially reversed 4μ8C-induced anomalies in tight junctions. Mechanistically, XBP1 inhibition resulted in increased intracellular Ca2+ concentration, upregulation of RhoA expression, redistribution of F-actin, and tight junction damage, which was attenuated by Rho kinase inhibitor Y27632. In vivo, deletion of XBP1 in the RPE resulted in defective RPE tight junctions accompanied by increased VEGF expression. Conclusions Taken together, these results suggest a protective role of XBP1 in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization. PMID:27701635

  12. Effect of Acute Stress on Immune Cell Counts and the Expression of Tight Junction Proteins in the Duodenal Mucosa of Rats

    PubMed Central

    Lee, Hong Sub; Kim, Dong-Kyu; Kim, Young Bae

    2013-01-01

    Background/Aims Duodenal immune alterations have been reported in a subset of patients with functional dyspepsia (FD). The aim of this study was to investigate the effect of acute stress on immune cell counts and the expression of tight junction proteins in the duodenal mucosa. Methods Twenty-one male rats were divided into the following three experimental groups: 1) the nonstressed, control group, 2) the 2-hour-stressed group, and 3) the 4-hour-stressed group. Eosinophils, mast cells and CD4+ and CD8+ T lymphocytes in the duodenal mucosa were counted. The protein and mRNA expressions of occludin and zonula occludens-1 (ZO-1) were examined. Results Eosinophils, mast cells and CD8+ T lymphocyte counts did not differ between the stressed and control groups. The number of CD4+ T lymphocytes and the protein and mRNA expressions of occludin and ZO-1 were significantly lower in the 4-hour-stressed group compared with the control group. The plasma adrenocorticotrophic hormone and cortisol levels of the 4-hour-stressed group were significantly higher than those of the control group. Conclusions Acute stress reduces the number of CD4+ T lymphocytes and the expression of tight junction proteins in the duodenal mucosa, which might be associated with the duodenal immune alterations found in a subset of FD patients. PMID:23560155

  13. 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.

  14. Tight Junction Defects in Atopic Dermatitis

    PubMed Central

    De Benedetto, Anna; Rafaels, Nicholas M.; McGirt, Laura Y.; Ivanov, Andrei I.; Georas, Steve N.; Cheadle, Chris; Berger, Alan E.; Zhang, Kunzhong; Vidyasagar, Sadasivan; Yoshida, Takeshi; Boguniewicz, Mark; Hata, Tissa; Schneider, Lynda C.; Hanifin, Jon M.; Gallo, Richard L.; Novak, Natalija; Weidinger, Stephan; Beaty, Terri H.; Leung, Donald Y.; Barnes, Kathleen C.; Beck, Lisa A.

    2010-01-01

    Background Atopic dermatitis (AD) is characterized by dry skin and a hyperreactive immune response to allergens, two cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJ) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. Objective We evaluated the expression/function of the TJ protein, claudin-1 in epithelium from AD and nonatopic (NA) subjects and screened two American populations for SNPs in CLDN1. Methods Expression profiles of nonlesional epithelium from extrinsic AD, NA and psoriasis subjects were generated using Illumina’s BeadChips. Dysregulated intercellular proteins were validated by tissue staining and qPCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed using a knockdown approach in primary human keratinocytes (PHK). Twenty seven haplotype-tagging SNPs in CLDN1 were screened in two independent AD populations. Results We observed strikingly reduced expression of the TJ proteins claudin-1 and -23 only in AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with Th2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro, we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging single nucleotide polymorphisms revealed associations with AD in two North American populations. Conclusion Taken together, these data suggest that an impaired epidermal TJ is a novel feature of skin barrier dysfunction and immune dysregulation observed in AD, and that CLDN1 may be a new susceptibility gene in this disease. PMID:21163515

  15. Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

    PubMed Central

    Tang, Vivian W

    2006-01-01

    Background Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of ~100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown. Results A novel biochemical purification protocol has been developed to isolate tight junction protein complexes from cultured human epithelial cells. After identification of proteins by mass spectroscopy and fingerprint analysis, candidate proteins are scored and assessed individually. A simple algorithm has been devised to incorporate transmembrane domains and protein modification sites for scoring membrane proteins. Using this new scoring system, a total of 912 proteins have been identified. These 912 hits are analyzed using a bioinformatics approach to bin the hits in 4 categories: configuration, molecular function, cellular function, and specialized process. Prominent clusters of proteins related to the cytoskeleton, cell adhesion, and vesicular traffic have been identified. Weaker clusters of proteins associated with cell growth, cell migration, translation, and transcription are also found. However, the strongest clusters belong to synaptic proteins and signaling molecules. Localization studies of key components of synaptic transmission have confirmed the presence of both presynaptic and postsynaptic proteins at the tight junction domain. To correlate proteomics data with structure, the tight junction has been examined using electron microscopy. This has revealed many novel structures including end-on cytoskeletal attachments, vesicles fusing/budding at the tight junction membrane domain, secreted substances encased between the tight junction kisses, endocytosis of tight junction double membranes, satellite

  16. Role of tight junction proteins in gastroesophageal reflux disease

    PubMed Central

    2012-01-01

    Background Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD. Methods Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)]. Results Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and −2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD. Conclusions Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD. PMID:22994974

  17. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier

    PubMed Central

    Verma, Saguna; Lo, Yeung; Chapagain, Moti; Lum, Stephanie; Kumar, Mukesh; Gurjav, Ulziijargal; Luo, Haiyan; Nakatsuka, Austin; Nerurkar, Vivek R.

    2009-01-01

    Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via ‘Trojan horse’ mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology. PMID:19135695

  18. A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier

    PubMed Central

    Wong, Vivian; Gumbiner, Barry M.

    1997-01-01

    Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 μM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal. PMID:9015310

  19. Erp29 Attenuates Cigarette Smoke Extract–Induced Endoplasmic Reticulum Stress and Mitigates Tight Junction Damage in Retinal Pigment Epithelial Cells

    PubMed Central

    Huang, Chuangxin; Wang, Joshua J.; Jing, Guangjun; Li, Junhua; Jin, Chenjin; Yu, Qiang; Falkowski, Marek W.; Zhang, Sarah X.

    2015-01-01

    Purpose Endoplasmic reticulum protein 29 (ERp29) is a novel chaperone that was recently found decreased in human retinas with AMD. Herein, we examined the effect of ERp29 on cigarette smoke–induced RPE apoptosis and tight junction disruption. Methods Cultured human RPE (HRPE) cells (ARPE-19) or mouse RPE eyecup explants were exposed to cigarette smoke extract (CSE) for short (up to 24 hours) or long (up to 3 weeks) periods. Expression of ERp29 was up- and downregulated by adenovirus and siRNA, respectively. Endoplasmic reticulum stress markers, apoptosis, and cell death, the expression and distribution of tight junction protein ZO-1, transepithelial electrical resistance (TEER), and F-actin expression were examined. Results Endoplasmic reticulum protein 29 was significantly increased by short-term exposure to CSE in ARPE-19 cells or eyecup explants but was reduced after 3-week exposure. Overexpression of ERp29 increased the levels of GRP78, p58IPK, and Nrf-2, while reducing p-eIF2α and C/EBP homologous protein (CHOP), and protected RPE cells from CSE-induced apoptosis. In contrast, knockdown of ERp29 decreased the levels of p58IPK and Nrf2, but increased p-eIF2α and CHOP and exacerbated CSE-triggered cell death. In addition, overexpression of ERp29 attenuated CSE-induced reduction in ZO-1 and enhanced the RPE barrier function, as measured by TEER. Knockdown of ERp29 decreased the level of ZO-1 protein. These effects were associated with changes in the expression of cytoskeleton F-actin. Conclusions Endoplasmic reticulum protein 29 attenuates CSE-induced ER stress and enhances cell viability and barrier integrity of RPE cells, and therefore may act as a protective mechanism for RPE survival and activity. PMID:26431474

  20. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions.

    PubMed

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the 'apical junctional complex-AJC' with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the 'Junctional Adhesion Molecules' (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

  1. 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.

  2. Anti-invasive activity of diallyl disulfide through tightening of tight junctions and inhibition of matrix metalloproteinase activities in LNCaP prostate cancer cells.

    PubMed

    Shin, Dong Yeok; Kim, Gi-Young; Kim, Jung-In; Yoon, Moo Kyoung; Kwon, Taeg Kyu; Lee, Su Jae; Choi, Young-Whan; Kang, Ho Sung; Yoo, Young Hyun; Choi, Yung Hyun

    2010-09-01

    Diallyl disulfide (DADS) is a major component of an oil-soluble allyl sulfide garlic (Allium sativum) derivative, which has been shown to exert a potential for anti-cancer activity. However, the biochemical mechanisms underlying DADS-induced anti-invasiveness and anti-metastasis have not been thoroughly studied. In this study, we investigated the effect of DADS on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in human prostate carcinoma LNCaP cells. Inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Additionally, immunoblotting results indicated that DADS repressed the levels of the claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)-2 and -9 in LNCaP cells were dose-dependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins. Although further studies are needed, the present study indicates that TJs and MMPs are critical targets of DADS-induced anti-invasiveness in human prostate cancer LNCaP cells.

  3. Deficiency of angulin-2/ILDR1, a tricellular tight junction-associated membrane protein, causes deafness with cochlear hair cell degeneration in mice.

    PubMed

    Higashi, Tomohito; Katsuno, Tatsuya; Kitajiri, Shin-Ichiro; Furuse, Mikio

    2015-01-01

    Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.

  4. Effect of diffusely adherent Escherichia coli strains isolated from diarrhoeal patients and healthy carriers on IL-8 secretion and tight junction barrier integrity of Caco-2 cells.

    PubMed

    Tanimoto, Yoshihiko; Arikawa, Kentaro; Nishikawa, Yoshikazu

    2013-03-15

    The pathogenesis of diffusely adherent Escherichia coli (DAEC) remains to be elucidated. Previously, we found that afimbrial adhesin gene (afa)-positive motile DAEC strains isolated from patients with diarrhoea induce high levels of IL-8 secretion in Caco-2 cells via toll-like receptor 5 (TLR-5), while non-motile strains did not. The aim of this study was to compare virulence properties, including the phylogenetic groups, afa subtypes, IL-8 secretion levels, and the effects on tight junctions, of DAEC strains isolated from healthy persons with those isolated from patients with diarrhoea. Induction of IL-8 secretion in Caco-2 cells was examined for a total of 36 afa-positive strains: 19 from diarrhoeal patients and 17 from healthy carriers. Irrespective of the source, all strains were classified into the phylogenetic group B2 or D, with the exception of two strains. All 7 motile strains isolated from diarrhoeal patients induced high levels of IL-8 secretion, while only 6 of 15 motile strains from healthy carriers induced IL-8 secretion to the same levels as the diarrhoeal strains. We speculated that additional virulence factors other than afa and motility cause the loosening of tight junctions that allows flagellin to reach TLR-5 located on the basolateral side of the epithelium. However, no differences in the TER and dextran permeability were observed between cells infected with diarrhoeal strains and those from healthy persons. Thus, diarrhoeagenic DAEC seems to possess additional factors, in addition to adhesin and flagellin, which can induce high IL-8 secretion.

  5. miR-200b inhibits TNF-α-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro.

    PubMed

    Shen, Yujie; Zhou, Min; Yan, Junkai; Gong, Zizhen; Xiao, Yongtao; Zhang, Cong; Du, Peng; Chen, Yingwei

    2017-02-01

    Inflammatory bowel diseases (IBDs) are chronic, inflammatory disorders of the gastrointestinal tract with unclear etiologies. Intestinal epithelial cells (IECs), containing crypt and villus enterocytes, occupy a critical position in the pathogenesis of IBDs and are a major producer of immunoregulatory cytokines and a key component of the intact epithelial barrier. Previously, we have reported that miR-200b is involved in the progression of IBDs and might maintain the integrity of the intestinal epithelial barrier via reducing the loss of enterocytes. In this study, we further investigated the impact of miR-200b on intestinal epithelial inflammation and tight junctions in two distinct differentiated states of Caco-2 cells after TNF-α treatment. We demonstrated that TNF-α-enhanced IL-8 expression was decreased by microRNA (miR)-200b in undifferentiated IECs. Simultaneously, miR-200b could alleviate TNF-α-induced tight junction (TJ) disruption in well-differentiated IECs by reducing the reduction in the transepithelial electrical resistance (TEER), inhibiting the increase in paracellular permeability, and preventing the morphological redistribution of the TJ proteins claudin 1 and ZO-1. The expression levels of the JNK/c-Jun/AP-1 and myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) pathways were attenuated in undifferentiated and differentiated enterocytes, respectively. Furthermore, a dual-luciferase reporter gene detection system provided direct evidence that c-Jun and MLCK were the specific targets of miR-200b. Collectively, our results highlighted that miR-200b played a positive role in IECs via suppressing intestinal epithelial IL-8 secretion and attenuating TJ damage in vitro, which suggested that miR-200b might be a promising strategy for IBD therapy.

  6. Distribution of Tight Junction Proteins in Adult Human Salivary Glands

    PubMed Central

    Maria, Ola M.; Kim, Jung-Wan Martin; Gerstenhaber, Jonathan A.; Baum, Bruce J.; Tran, Simon D.

    2008-01-01

    Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008) PMID:18765838

  7. ZO-2 silencing in epithelial cells perturbs the gate and fence function of tight junctions and leads to an atypical monolayer architecture.

    PubMed

    Hernandez, Sandra; Chavez Munguia, Bibiana; Gonzalez-Mariscal, Lorenza

    2007-05-01

    ZO-2 is a tight junction (TJ) protein that shuttles between the plasma membrane and the nucleus. ZO-2 contains several protein binding sites that allow it to function as a scaffold that clusters integral, adaptor and signaling proteins. To gain insight into the role of ZO-2 in epithelial cells, ZO-2 was silenced in MDCK cells with small interference RNA (siRNA). ZO-2 silencing triggered: (A) changes in the gate function of the TJ, determined by an increase in dextran flow through the paracellular route of mature monolayers and achievement of lower transepithelial electrical resistance values upon TJ de novo formation; (B) changes in the fence function of the TJ manifested by a non-polarized distribution of E-cadherin on the plasma membrane; (C) altered expression of TJ and adherens junction proteins, determined by a decreased amount of occludin and E-cadherin in mature monolayers and a delayed arrival to the plasma membrane of ZO-1, occludin and E-cadherin during a calcium switch assay; and (D) an atypical monolayer architecture characterized by the appearance of widened intercellular spaces, multistratification of regions in the culture and an altered pattern of actin at the cellular borders.

  8. Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells

    PubMed Central

    2010-01-01

    Background Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells. Results Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization. Conclusion Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and subcellular distribution suggests

  9. Tight junction disruption: Helicobacter pylori and dysregulation of the gastric mucosal barrier

    PubMed Central

    Caron, Tyler J; Scott, Kathleen E; Fox, James G; Hagen, Susan J

    2015-01-01

    Long-term chronic infection with Helicobacter pylori (H. pylori) is a risk factor for gastric cancer development. In the multi-step process that leads to gastric cancer, tight junction dysfunction is thought to occur and serve as a risk factor by permitting the permeation of luminal contents across an otherwise tight mucosa. Mechanisms that regulate tight junction function and structure in the normal stomach, or dysfunction in the infected stomach, however, are largely unknown. Although conventional tight junction components are expressed in gastric epithelial cells, claudins regulate paracellular permeability and are likely the target of inflammation or H. pylori itself. There are 27 different claudin molecules, each with unique properties that render the mucosa an intact barrier that is permselective in a way that is consistent with cell physiology. Understanding the architecture of tight junctions in the normal stomach and then changes that occur during infection is important but challenging, because most of the reports that catalog claudin expression in gastric cancer pathogenesis are contradictory. Furthermore, the role of H. pylori virulence factors, such as cytotoxin-associated gene A and vacoulating cytotoxin, in regulating tight junction dysfunction during infection is inconsistent in different gastric cell lines and in vivo, likely because non-gastric epithelial cell cultures were initially used to unravel the details of their effects on the stomach. Hampering further study, as well, is the relative lack of cultured cell models that have tight junction claudins that are consistent with native tissues. This summary will review the current state of knowledge about gastric tight junctions, normally and in H. pylori infection, and make predictions about the consequences of claudin reorganization during H. pylori infection. PMID:26523106

  10. Blood-brain barrier tight junction permeability and ischemic stroke.

    PubMed

    Sandoval, Karin E; Witt, Ken A

    2008-11-01

    The blood-brain barrier (BBB) is formed by the endothelial cells of cerebral microvessels, providing a dynamic interface between the peripheral circulation and the central nervous system. The tight junctions (TJs) between the endothelial cells serve to restrict blood-borne substances from entering the brain. Under ischemic stroke conditions decreased BBB TJ integrity results in increased paracellular permeability, directly contributing to cerebral vasogenic edema, hemorrhagic transformation, and increased mortality. This loss of TJ integrity occurs in a phasic manner, which is contingent on several interdependent mechanisms (ionic dysregulation, inflammation, oxidative and nitrosative stress, enzymatic activity, and angiogenesis). Understanding the inter-relation of these mechanisms is critical for the development of new therapies. This review focuses on those aspects of ischemic stroke impacting BBB TJ integrity and the principle regulatory pathways, respective to the phases of paracellular permeability.

  11. Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

    SciTech Connect

    Li, Zhao; Jin, Zhu-Qiu

    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 cardiac 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

  12. Ferulate protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in tert-butyl hydroperoxide-induced Caco-2 cells.

    PubMed

    Kim, Hyun Jung; Lee, Eun Kyeong; Park, Min Hi; Ha, Young Mi; Jung, Kyung Jin; Kim, Min-Sun; Kim, Mi Kyung; Yu, Byung Pal; Chung, Hae Young

    2013-03-01

    Epithelial barrier function is determined by both transcellular and paracellular permeability, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. We investigated the protective effects of ferulate on epithelial barrier integrity by examining permeability, TJ protein expression, and apoptosis in Caco-2 cells treated with tert-butyl hydroperoxide (t-BHP), a strong reactive species inducer. Caco-2 cells pretreated with ferulate (5 or 15 μM) were exposed to t-BHP (100 μM), and ferulate suppressed the t-BHP-mediated increases in reactive species and epithelial permeability in Caco-2 cells. Moreover, ferulate inhibited epithelial cell leakage induced by t-BHP, which was accompanied by decreased expression of the TJ proteins zonula occludens-1 and occludin. In addition, pretreatment with ferulate markedly protected cells against t-BHP-induced apoptosis, as evidenced by decreased nuclear condensation, cytochrome c release, and caspase-3 cleavage and an increased Bax/Bcl-2 ratio. These results suggest that ferulate protects the epithelial barrier of Caco-2 cells against oxidative stress, which results in increased epithelial permeability, decreased TJ protein expression, and increased apoptosis. The most significant finding of our study is the demonstration of protective, ferulate-mediated antioxidant effects on barrier integrity, with a particular focus on intracellular molecular mechanisms.

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

    PubMed

    Guignot, Julie; Chaplais, Cécile; Coconnier-Polter, Marie-Hélène; Servin, Alain L

    2007-01-01

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

  14. Irinotecan disrupts tight junction proteins within the gut

    PubMed Central

    Wardill, Hannah R; Bowen, Joanne M; Al-Dasooqi, Noor; Sultani, Masooma; Bateman, Emma; Stansborough, Romany; Shirren, Joseph; Gibson, Rachel J

    2014-01-01

    Chemotherapy for cancer causes significant gut toxicity, leading to severe clinical manifestations and an increased economic burden. Despite much research, many of the underlying mechanisms remain poorly understood hindering effective treatment options. Recently there has been renewed interest in the role tight junctions play in the pathogenesis of chemotherapy-induced gut toxicity. To delineate the underlying mechanisms of chemotherapy-induced gut toxicity, this study aimed to quantify the molecular changes in key tight junction proteins, ZO-1, claudin-1, and occludin, using a well-established preclinical model of gut toxicity. Female tumor-bearing dark agouti rats received irinotecan or vehicle control and were assessed for validated parameters of gut toxicity including diarrhea and weight loss. Rats were killed at 6, 24, 48, 72, 96, and 120 h post-chemotherapy. Tight junction protein and mRNA expression in the small and large intestines were assessed using semi-quantitative immunohistochemistry and RT-PCR. Significant changes in protein expression of tight junction proteins were seen in both the jejunum and colon, correlating with key histological changes and clinical features. mRNA levels of claudin-1 were significantly decreased early after irinotecan in the small and large intestines. ZO-1 and occludin mRNA levels remained stable across the time-course of gut toxicity. Findings strongly suggest irinotecan causes tight junction defects which lead to mucosal barrier dysfunction and the development of diarrhea. Detailed research is now warranted to investigate posttranslational regulation of tight junction proteins to delineate the underlying pathophysiology of gut toxicity and identify future therapeutic targets. PMID:24316664

  15. Claudin-21 Has a Paracellular Channel Role at Tight Junctions.

    PubMed

    Tanaka, Hiroo; Yamamoto, Yasuko; Kashihara, Hiroka; Yamazaki, Yuji; Tani, Kazutoshi; Fujiyoshi, Yoshinori; Mineta, Katsuhiko; Takeuchi, Kosei; Tamura, Atsushi; Tsukita, Sachiko

    2016-01-04

    Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.

  16. Trends in drug delivery through tissue barriers containing tight junctions

    PubMed Central

    Tscheik, Christian; Blasig, Ingolf E.; Winkler, Lars

    2013-01-01

    A limitation in the uptake of many drugs is the restricted permeation through tissue barriers. There are two general ways to cross barriers formed by cell layers: by transcytosis or by diffusion through the intercellular space. In the latter, tight junctions (TJs) play the decisive role in the regulation of the barrier permeability. Thus, transient modulation of TJs is a potent strategy to improve drug delivery. There have been extensive studies on surfactant-like absorption enhancers. One of the most effective enhancers found is sodium caprate. However, this modulates TJs in an unspecific fashion. A novel approach would be the specific modulation of TJ-associated marvel proteins and claudins, which are the main structural components of the TJs. Recent studies have identified synthetic peptidomimetics and RNA interference techniques to downregulate the expression of targeted TJ proteins. This review summarizes current progress and discusses the impact on TJs' barrier function. PMID:24665392

  17. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    PubMed

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati

    2004-05-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  18. Clostridium perfringens Enterotoxin Elicits Rapid and Specific Cytolysis of Breast Carcinoma Cells Mediated through Tight Junction Proteins Claudin 3 and 4

    PubMed Central

    Kominsky, Scott L.; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G.; Weitzman, Sigmund A.; Argani, Pedram; Sukumar, Saraswati

    2004-01-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy. PMID:15111309

  19. p38 Mitogen-activated protein kinase and c-Jun NH2-terminal protein kinase regulate the accumulation of a tight junction protein, ZO-1, in cell-cell contacts in HaCaT cells.

    PubMed

    Minakami, Masahiko; Kitagawa, Norio; Iida, Hiroshi; Anan, Hisashi; Inai, Tetsuichiro

    2015-02-01

    To investigate the involvement of stress-activated protein kinases, JNK and p38 MAPK, in the assembly of tight junctions in keratinocytes, we treated HaCaT cells with various combinations of SP600125 (an inhibitor of JNK), SB202190 (an inhibitor of p38 MAPK) and anisomycin (an activator of both JNK and p38 MAPK) and examined the localization of ZO-1, an undercoat constitutive protein of the tight junction. Short-term (8h) incubation with SP600125, SB202190 or anisomycin induced the accumulation of ZO-1 in the cell-cell contacts, with reduced ZO-1 staining in the cytoplasm, while only long-term (24h) incubation with SP600125 induced the accumulation of ZO-1. SP600125, SB202190 or SP600125 plus SB202190 treatment induced thin linear staining for ZO-1 in the cell-cell contacts. Anisomycin treatment induced thick and irregular linear staining for ZO-1, while anisomycin plus SP600125 treatment induced zipper-like staining for ZO-1. Anisomycin plus SB202190 treatment or anisomycin plus both SP600125 and SB202190 treatment for 8h failed to lead to the accumulation of ZO-1 in cell-cell contacts, but induced thin linear staining with several gaps 16 h after removal of these agents. These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period.

  20. 21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions

    PubMed Central

    Rocha, Sayonarah C.; Pessoa, Marco T. C.; Neves, Luiza D. R.; Alves, Silmara L. G.; Silva, Luciana M.; Santos, Herica L.; Oliveira, Soraya M. F.; Taranto, Alex G.; Comar, Moacyr; Gomes, Isabella V.; Santos, Fabio V.; Paixão, Natasha; Quintas, Luis E. M.; Noël, François; Pereira, Antonio F.; Tessis, Ana C. S. C.; Gomes, Natalia L. S.; Moreira, Otacilio C.; Rincon-Heredia, Ruth; Varotti, Fernando P.; Blanco, Gustavo; Villar, Jose A. F. P.; Contreras, Rubén G.; Barbosa, Leandro A.

    2014-01-01

    Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions. PMID:25290152

  1. 21-Benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na,K-ATPase and epithelial tight junctions.

    PubMed

    Rocha, Sayonarah C; Pessoa, Marco T C; Neves, Luiza D R; Alves, Silmara L G; Silva, Luciana M; Santos, Herica L; Oliveira, Soraya M F; Taranto, Alex G; Comar, Moacyr; Gomes, Isabella V; Santos, Fabio V; Paixão, Natasha; Quintas, Luis E M; Noël, François; Pereira, Antonio F; Tessis, Ana C S C; Gomes, Natalia L S; Moreira, Otacilio C; Rincon-Heredia, Ruth; Varotti, Fernando P; Blanco, Gustavo; Villar, Jose A F P; Contreras, Rubén G; Barbosa, Leandro A

    2014-01-01

    Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.

  2. Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

    PubMed

    Stremmel, Wolfgang; Staffer, Simone; Gan-Schreier, Hongying; Wannhoff, Andreas; Bach, Margund; Gauss, Annika

    2016-09-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.

  3. 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.

  4. Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier

    PubMed Central

    Bon, Fabienne; L’Ollivier, Coralie; Laue, Michael; Holland, Gudrun; Bonnin, Alain; Dalle, Frederic

    2016-01-01

    C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ) formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin), we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ. PMID:26933885

  5. 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

  6. 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.

  7. Heterogeneity of tight junction morphology in extrapulmonary and intrapulmonary airways of the rat.

    PubMed

    Schneeberger, E E

    1980-10-01

    In the present study morphology of tight junctions was related to the various cell types lining extrapulmonary and intrapulmonary airways of the rat. Freeze fracture replicas were prepared from extrapulmonary airway epithelium derived from the cartilagenous and membranous sides of upper, middle, and lower thirds of the trachea. Intrapulmonary airway epithelium was obtained from airways less than 1 mm in diameter. Tight junction fibrils on the P fracture face were organized into three types of patterns. Type 1: parallel sparsely interconnected lumenal fibrils with large ablumenal fibril loops. Type 2: richly interconnected lumenal fibrils with large ablumenal fibril loops. Type 3: narrow network of interconnected fibrils. On the E fracture face complementary grooves were organized in a similar pattern. Ciliated cells on both sides and all levels of the trachea were associated with type 1 junctions. In intrapulmonary airways, however, the junctional pattern of ciliated cells changed to type 2. Brush cells at all levels of the airways were bounded by type 2 and occasionally by type 1 junctions. Secretory cell junctions displayed the following patterns: Mucous cells were bounded solely by type 3, serous cells by either types 2 or 3, and Clara cells predominantly by type 2. Cells tentatively identified as intermediate cells displayed all three junctional patterns. The number of parallel fibrils comprising tight junctions was higher in extrapulmonary as compared to intrapulmonary airways. No difference was seen in the various locations sampled in the trachea. Gap junctions were observed between secretory cells of extrapulmonary but not intrapulmonary airways. These observations are discussed in relation to current physiologic data.

  8. Arsenic downregulates tight junction claudin proteins through p38 and NF-κB in intestinal epithelial cell line, HT-29.

    PubMed

    Jeong, Chang Hee; Seok, Jin Sil; Petriello, Michael C; Han, Sung Gu

    2017-03-15

    Arsenic is a naturally occurring metalloid that often is found in foods and drinking water. Human exposure to arsenic is associated with the development of gastrointestinal problems such as fluid loss, diarrhea and gastritis. Arsenic is also known to induce toxic responses including oxidative stress in cells of the gastrointestinal track. Tight junctions (TJs) regulate paracellular permeability and play a barrier role by inhibiting the movement of water, solutes and microorganisms in the paracellular space. Since oxidative stress and TJ damage are known to be associated, we examined whether arsenic produces TJ damage such as downregulation of claudins in the human colorectal cell line, HT-29. To confirm the importance of oxidative stress in arsenic-induced TJ damage, effects of the antioxidant compound (e.g., N-acetylcysteine (NAC)) were also determined in cells. HT-29 cells were treated with arsenic trioxide (40μM, 12h) to observe the modified expression of TJ proteins. Arsenic decreased expression of TJ proteins (i.e., claudin-1 and claudin-5) and transepithelial electrical resistance (TEER) whereas pretreatment of NAC (5-10mM, 1h) attenuated the observed claudins downregulation and TEER. Arsenic treatment produced cellular oxidative stress via superoxide generation and lowering glutathione (GSH) levels, while NAC restored cellular GSH levels and decreased oxidative stress. Arsenic increased phosphorylation of p38 and nuclear translocation of nuclear factor-kappa B (NF-κB) p65, while NAC attenuated these intracellular events. Results demonstrated that arsenic can damage intestinal epithelial cells by proinflammatory process (oxidative stress, p38 and NF-κB) which resulted in the downregulation of claudins and NAC can protect intestinal TJs from arsenic toxicity.

  9. Emerging Multifunctional Roles of Claudin Tight Junction Proteins in Bone

    PubMed Central

    Alshbool, Fatima. Z.

    2014-01-01

    The imbalance between bone formation and resorption during bone remodeling has been documented to be a major factor in the pathogenesis of osteoporosis. Recent evidence suggests a significant role for the tight junction proteins, Claudins (Cldns), in the regulation of bone remodeling processes. In terms of function, whereas Cldns act “canonically” as key determinants of paracellular permeability, there is considerable recent evidence to suggest that Cldns also participate in cell signaling, ie, a “noncanonical function”. To this end, Cldns have been shown to regulate cell proliferation, differentiation, and gene expression in a variety of cell types. The present review will discuss Cldns' structure, their expression profile, regulation of expression, and their canonical and non- canonical functions in general with special emphasis on bone cells. In order to shed light on the noncanonical functions of Cldns in bone, we will highlight the role of Cldn-18 in regulating bone resorption and osteoclast differentiation. Collectively, we hope to provide a framework for guiding future research on understanding how Cldns modulate osteoblast and osteoclast function and overall bone homeostasis. Such studies should provide valuable insights into the pathogenesis of osteoporosis, and may highlight Cldns as novel targets for the diagnosis and therapeutic management of osteoporosis. PMID:24758302

  10. Emerging multifunctional roles of Claudin tight junction proteins in bone.

    PubMed

    Alshbool, Fatima Z; Mohan, Subburaman

    2014-07-01

    The imbalance between bone formation and resorption during bone remodeling has been documented to be a major factor in the pathogenesis of osteoporosis. Recent evidence suggests a significant role for the tight junction proteins, Claudins (Cldns), in the regulation of bone remodeling processes. In terms of function, whereas Cldns act "canonically" as key determinants of paracellular permeability, there is considerable recent evidence to suggest that Cldns also participate in cell signaling, ie, a "noncanonical function". To this end, Cldns have been shown to regulate cell proliferation, differentiation, and gene expression in a variety of cell types. The present review will discuss Cldns' structure, their expression profile, regulation of expression, and their canonical and non- canonical functions in general with special emphasis on bone cells. In order to shed light on the noncanonical functions of Cldns in bone, we will highlight the role of Cldn-18 in regulating bone resorption and osteoclast differentiation. Collectively, we hope to provide a framework for guiding future research on understanding how Cldns modulate osteoblast and osteoclast function and overall bone homeostasis. Such studies should provide valuable insights into the pathogenesis of osteoporosis, and may highlight Cldns as novel targets for the diagnosis and therapeutic management of osteoporosis.

  11. The role of tight junctions in mammary gland function.

    PubMed

    Stelwagen, Kerst; Singh, Kuljeet

    2014-03-01

    Tight junctions (TJ) are cellular structures that facilitate cell-cell communication and are important in maintaining the three-dimensional structure of epithelia. It is only during the last two decades that the molecular make-up of TJ is becoming unravelled, with two major transmembrane-spanning structural protein families, called occludin and claudins, being the true constituents of the TJ. These TJ proteins are linked via specific scaffolding proteins to the cell's cytoskeleton. In the mammary gland TJ between adjacent secretory epithelial cells are formed during lactogenesis and are instrumental in establishing and maintaining milk synthesis and secretion, whereas TJ integrity is compromised during mammary involution and also as result of mastitis and periods of mammary inflamation (including mastitis). They prevent the paracellular transport of ions and small molecules between the blood and milk compartments. Formation of intact TJ at the start of lactation is important for the establishment of the lactation. Conversely, loss of TJ integrity has been linked to reduced milk secretion and mammary function and increased paracellular transport of blood components into the milk and vice versa. In addition to acting as a paracellular barrier, the TJ is increasingly linked to playing an active role in intracellular signalling. This review focusses on the role of TJ in mammary function of the normal, non-malignant mammary gland, predominantly in ruminants, the major dairy producing species.

  12. Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin, Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation

    PubMed Central

    Ozawa, Masayuki; Kobayashi, Wakako

    2014-01-01

    The downregulation of E-cadherin function has fundamental consequences with respect to cancer progression, and occurs as part of the epithelial–mesenchymal transition (EMT). In this study, we show that the expression of the Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domain in cells inhibited the cell surface localization of endogenous E-cadherin, leading to morphological changes, the inhibition of junctional assembly and cell dissociation. These changes were associated with increased cell migration, but were not accompanied by the down-regulation of epithelial markers and up-regulation of mesenchymal markers. Thus, these changes cannot be classified as EMT. The cadherin cytoplasmic domain interacted with β-catenin or plakoglobin, reducing the levels of β-catenin or plakoglobin associated with E-cadherin, and raising the possibility that β-catenin and plakoglobin sequestration by these constructs induced E-cadherin intracellular localization. Accordingly, a cytoplasmic domain construct bearing mutations that weakened the interactions with β-catenin or plakoglobin did not impair junction formation and adhesion, indicating that the interaction with β-catenin or plakoglobin was essential to the potential of the constructs. E-cadherin–α-catenin chimeras that did not require β-catenin or plakoglobin for their cell surface transport restored cell–cell adhesion and junction formation. PMID:25121615

  13. Rapid method of quantification of tight-junction organization using image analysis.

    PubMed

    Terryn, Christine; Sellami, Mehdi; Fichel, Caroline; Diebold, Marie-Danielle; Gangloff, Sophie; Le Naour, Richard; Polette, Myriam; Zahm, Jean-Marie

    2013-02-01

    The spatial organization of proteins in a cell population or in tissues is an important parameter to study the functionality of biological specimens. In this article, we have focused on tight junctions which form network-like features in immunofluorescence microscopy images. Usually, the organization or disorganization of tight junctions is noticed qualitatively. The aim of this article is to present a simple method to quantify the organization level of tight junction network using image analysis with a dedicated macro developed with Image J software. The method has been validated with simulated images displaying regular decrease of network organization. Then, the macro has been applied to immunofluorescence microscopy images of cells in culture and of tissue sections.

  14. PLEKHA7 modulates epithelial tight junction barrier function

    PubMed Central

    Paschoud, Serge; Jond, Lionel; Guerrera, Diego; Citi, Sandra

    2014-01-01

    PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms. PMID:24843844

  15. ILDR1 null mice, a model of human deafness DFNB42, show structural aberrations of tricellular tight junctions and degeneration of auditory hair cells.

    PubMed

    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

    2015-02-01

    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.

  16. Tight junction regulates epidermal calcium ion gradient and differentiation

    SciTech Connect

    Kurasawa, Masumi; Maeda, Tetsuo; Oba, Ai; Yamamoto, Takuya; Sasaki, Hiroyuki

    2011-03-25

    Research highlights: {yields} We disrupted epidermal tight junction barrier in reconstructed epidermis. {yields} It altered Ca{sup 2+} distribution and consequentially differentiation state as well. {yields} Tight junction should affect epidermal homeostasis by maintaining Ca{sup 2+} gradient. -- Abstract: It is well known that calcium ions (Ca{sup 2+}) induce keratinocyte differentiation. Ca{sup 2+} distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca{sup 2+} gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca{sup 2+} gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca{sup 2+} flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca{sup 2+} gradient.

  17. Gonadotropins regulate rat testicular tight junctions in vivo.

    PubMed

    McCabe, Mark J; Tarulli, Gerard A; Meachem, Sarah J; Robertson, David M; Smooker, Peter M; Stanton, Peter G

    2010-06-01

    Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG +/- FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

  18. Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo

    PubMed Central

    McCabe, Mark J.; Tarulli, Gerard A.; Meachem, Sarah J.; Robertson, David M.; Smooker, Peter M.; Stanton, Peter G.

    2010-01-01

    Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG ± FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs. PMID:20357222

  19. 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.

  20. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions

    PubMed Central

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the ‘apical junctional complex—AJC’ with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the ‘Junctional Adhesion Molecules’ (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers. PMID:25610754

  1. Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells

    PubMed Central

    Bleau, Christian; Filliol, Aveline; Samson, Michel

    2015-01-01

    CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion. PMID:26202229

  2. Rapid remodeling of tight junctions during paracellular diapedesis in a human model of the blood-brain barrier.

    PubMed

    Winger, Ryan C; Koblinski, Jennifer E; Kanda, Takashi; Ransohoff, Richard M; Muller, William A

    2014-09-01

    Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.

  3. Nanoagonist-mediated endothelial tight junction opening: A strategy for safely increasing brain drug delivery in mice.

    PubMed

    Gao, Xihui; Wang, Yuan-Cheng; Liu, Yikang; Yue, Qi; Liu, Zining; Ke, Mengjing; Zhao, Shengyuan; Li, Cong

    2017-04-01

    Even though opening endothelial tight junctions is an efficient way to up-regulate brain drug delivery, the extravasation of blood-borne components from the compromised tight junctions can result in adverse consequences such as edema and neuronal injuries. In this work, we developed a nanoagonist that temporarily opened tight junctions by signaling adenosine 2A receptor, a type of G protein-coupled receptor expressed on brain capillary endothelial cells. Magnetic resonance imaging demonstrated remarkable blood-brain barrier permeability enhancements and significantly increased brain uptakes of both small molecular and macromolecular paramagnetic agents after nanoagonist administration. Gamma ray imaging and transmission electron microscope observed tight junction opening followed by spontaneous recovery after nanoagonist treatment. Immunofluorescence staining showed the unspoiled basal membrane, pericytes and astrocyte endfeet that enwrapped the vascular endothelium. Importantly, edema, apoptosis and neuronal injuries observed after hypertonic agent mediated tight junction-opening were not observed after nanoagonist intervention. The uncompromised neurovascular units may prevent the leakage of blood-borne constituents into brain parenchyma and accelerate tight junction recovery. Considering blood-brain barrier impermeability is a major obstacle in the treatment of central nervous system diseases, nanoagonist-mediated tight junction opening provides a promising strategy to enhance brain drug delivery with minimized adverse effects.

  4. Gastrointestinal mucositis: the role of MMP-tight junction interactions in tissue injury.

    PubMed

    Al-Dasooqi, Noor; Wardill, Hannah R; Gibson, Rachel J

    2014-07-01

    Chemotherapy for cancer causes significant gut toxicity known as mucositis. The pathogenesis of mucositis is ill defined. Recent clinical research guidelines have highlighted epithelial junctional complexes as emerging targets within mucositis research. Given the robust biological evidence linking tight junctions and matrix metalloproteinases, key mediators of mucositis, tight junction proteins have received significant attention. Despite this, the link between tight junctions, matrix metalloproteinases and mucositis development is yet to be established. This critical review therefore aims to describe the role of matrix metalloproteinases in mucositis, and how matrix metalloproteinase-dependent tight junction disruption may contribute to the pathobiology of mucositis.

  5. Diffusion of nonelectrolytes in the canine trachea: effect of tight junction.

    PubMed

    George, S C; Babb, A L; Deffebach, M E; Hlastala, M P

    1996-05-01

    We recently demonstrated through theoretical modeling that the exhaled ethanol (EtOH) profile from humans is consistent with a molecular diffusion coefficient (cm2/s) in the bronchial mucosa (Dti) that is only 8% of the diffusion coefficient in water (Dw; J. Appl. Physiol. 75: 2439-2449, 1993). Because of the small oil-water partition coefficient (lambda o:w) of EtOH (lambda o:w = 0.074), the reduced diffusion coefficient may be due, in part, to the epithelial tight junction in the paracellular pathway. We hypothesized that opening the tight junction would open an aqueous pathway and increase the diffusion coefficient of small (mol wt < 100) hydrophilic compounds. We mounted the mucosa from the membranous canine trachea in an Ussing-type diffusion cell and measured the diffusion coefficient of 2-ethoxyethanol (2-Ethx; lambda o:w = 0.042), EtOH, and methyl ethyl ketone (MEK; lambda o:w = 1.04) in the presence and absence of the epithelial tight junction. The tight junction was opened using a phosphate-buffered saline free of Ca2+ and Mg2+ with 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and its integrity was assessed by measuring the transepithelial electrical resistance. Dti/Dw in the presence of Ca2+ and Mg2+ was 0.39, 0.34, and 0.39 for 2-Ethx, EtOH, and MEK, respectively, and increased 24.6, 11.7, and 1.11% in the absence of Ca2+ and Mg2+. We conclude that the effect of the tight junction on Dti increases with increasing water solubility but can account for only a small portion of the reduced Dti of EtOH as predicted by exhaled profiles.

  6. 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.

  7. 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

  8. Formation of tight junctions between neighboring podocytes is an early ultrastructural feature in experimental crescentic glomerulonephritis

    PubMed Central

    Succar, Lena; Boadle, Ross A; Harris, David C; Rangan, Gopala K

    2016-01-01

    Purpose In crescentic glomerulonephritis (CGN), the development of cellular bridges between podocytes and parietal epithelial cells (PECs) triggers glomerular crescent formation. However, the sequential changes in glomerular ultrastructure in CGN are not well defined. This study investigated the time course of glomerular ultrastructure in experimental CGN. Methods Transmission electron microscopy (TEM) was performed using kidney samples from rats with nephrotoxic serum nephritis (NSN) from day 1 to day 14. Morphometric analysis was conducted on randomly selected glomeruli captured on TEM digital images. Results On day 1 of NSN, there was widespread formation of focal contacts between the cell bodies of neighboring podocytes, and tight junctions were evident at the site of cell-to-cell contact. This was confirmed by the increased expression of the tight junction molecule, zonula occludens-1 (ZO-1), which localized to the points of podocyte cell–cell body contact. On day 2, the interpodocyte distance decreased and the glomerular basement membrane thickness increased. Foot process effacement (FPE) was segmental on day 3 and diffuse by day 5, accompanied by the formation of podocyte cellular bridges with Bowman’s capsule, as confirmed by a decrease in podocyte-to-PEC distance. Fibrinoid necrosis and cellular crescents were evident in all glomeruli by days 7 and 14. In vitro, the exposure of podocytes to macrophage-conditioned media altered cellular morphology and caused an intracellular redistribution of ZO-1. Conclusion The formation of tight junctions between podocytes is an early ultrastructural abnormality in CGN, preceding FPE and podocyte bridge formation and occurring in response to inflammatory injury. Podocyte-to-podocyte tight junction formation may be a compensatory mechanism to maintain the integrity of the glomerular filtration barrier following severe endocapillary injury. PMID:27920570

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

    PubMed Central

    Takeichi, Masatoshi

    2006-01-01

    On December 11, 2005, Shoichiro Tsukita died at the young age of 52, after 14 months of treatment for cancer. Early in his career, Tsukita succeeded in isolating and purifying the adherens junction with his wife Sachiko, an accomplishment that he followed up with an impressive series of discoveries of cell adhesion and cytoskeletal molecules, including what may have been his greatest contribution to the field, the identification of occludin and the claudin family of molecules, which were watershed discoveries in the study of the molecular nature of tight junctions. PMID:16449186

  10. They Must Hold Tight: Junction Proteins, Microbiota And Immunity In Intestinal Mucosa.

    PubMed

    Castoldi, Angela; Favero de Aguiar, Cristhiane; Moraes-Vieira, Pedro Manoel; Olsen Saraiva Câmara, Niels

    2015-01-01

    Homeostasis of the immune system depends on several factors. The gastrointestinal tract plays an important role in maintaining our immune system. With this aim, the intestinal immune system interacts with epithelial barrier molecules, especially tight junction proteins, that are key molecules involved in controlling paracellular permeability to increase the protection barrier against external antigens or possibly to respond to commensal microorganisms. During intestinal inflammatory diseases, the expression of innate immune receptors in intestinal epithelial cells and infiltration of immune cells are related, but it is still unclear how the immune system induces modulation of paracellular permeability. In this review, we provide an overview of the understanding of how the immune system modulates the expression of tight junctions to maintain the mucosal immune system.

  11. Group A Streptococcus exploits human plasminogen for bacterial translocation across epithelial barrier via tricellular tight junctions

    PubMed Central

    Sumitomo, Tomoko; Nakata, Masanobu; Higashino, Miharu; Yamaguchi, Masaya; Kawabata, Shigetada

    2016-01-01

    Group A Streptococcus (GAS) is a human-specific pathogen responsible for local suppurative and life-threatening invasive systemic diseases. Interaction of GAS with human plasminogen (PLG) is a salient characteristic for promoting their systemic dissemination. In the present study, a serotype M28 strain was found predominantly localized in tricellular tight junctions of epithelial cells cultured in the presence of PLG. Several lines of evidence indicated that interaction of PLG with tricellulin, a major component of tricellular tight junctions, is crucial for bacterial localization. A site-directed mutagenesis approach revealed that lysine residues at positions 217 and 252 within the extracellular loop of tricellulin play important roles in PLG-binding activity. Additionally, we demonstrated that PLG functions as a molecular bridge between tricellulin and streptococcal surface enolase (SEN). The wild type strain efficiently translocated across the epithelial monolayer, accompanied by cleavage of transmembrane junctional proteins. In contrast, amino acid substitutions in the PLG-binding motif of SEN markedly compromised those activities. Notably, the interaction of PLG with SEN was dependent on PLG species specificity, which influenced the efficiency of bacterial penetration. Our findings provide insight into the mechanism by which GAS exploits host PLG for acceleration of bacterial invasion into deeper tissues via tricellular tight junctions. PMID:26822058

  12. The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells.

    PubMed

    Cavic, Milena; Grozdanovic, Milica M; Bajic, Aleksandar; Jankovic, Radmila; Andjus, Pavle R; Gavrovic-Jankulovic, Marija

    2014-10-01

    Actinidin, a kiwifruit cysteine protease, is a marker allergen for genuine sensitization to this food allergen source. Inhalatory cysteine proteases have the capacity for disruption of tight junctions (TJs) enhancing the permeability of the bronchial epithelium. No such properties have been reported for allergenic food proteases so far. The aim was to determine the effect of actinidin on the integrity of T84 monolayers by evaluating its action on the TJ protein occludin. Immunoblot and immunofluorescence were employed for the detection of occludin protein alterations. Gene expression was evaluated by RT-PCR. Breach of occludin network was assessed by measuring transepithelial resistance, blue dextran leakage and passage of allergens from the apical to basolateral compartment. Actinidin exerted direct proteolytic cleavage of occludin; no alteration of occludin gene expression was detected. There was a reduction of occludin staining upon actinidin treatment as a consequence of its degradation and dispersion within the membrane. There was an increase in permeability of the T84 monolayer resulting in reduced transepithelial resistance, blue dextran leakage and passage of allergens actinidin and thaumatin-like protein from the apical to basolateral compartment. Opening of TJs by actinidin may increase intestinal permeability and contribute to the process of sensitization in kiwifruit allergy.

  13. Tongxinluo Regulates Expression of Tight Junction Proteins and Alleviates Endothelial Cell Monolayer Hyperpermeability via ERK-1/2 Signaling Pathway in Oxidized Low-Density Lipoprotein-Induced Human Umbilical Vein Endothelial Cells

    PubMed Central

    2017-01-01

    Vascular hyperpermeability resulting from distortion of endothelial junctions is associated with a number of cardiovascular diseases. Endothelial tight junction regulates the paracellular permeability of macromolecules, a function of Human Umbilical Vein Endothelial Cells (HUVEC) monolayers that can be regulated by oxidized Low-density Lipoprotein (ox-LDL). However, the understanding of drug regulation of vascular hyperpermeability is so far limited. This study thus aimed to investigate the role of Tongxinluo (TXL) in the maintenance of the vascular endothelial paracellular permeability. Here, changes in permeability were determined by measuring the paracellular flux of FITC-dextran 40000 (FD40), while protein expression and intercellular distribution were examined by western blot and immunofluorescence assay, respectively. We found that TXL alleviated the ox-LDL-induced increase in flux of FD40 and then reduced the hyperpermeability. Moreover, ox-LDL-induced disruptions of ZO-1, occludin, and claudin1 were also restored. This is via the activation of ERK1/2 in the vascular endothelial cells. Our results provide insights into the molecular mechanism by which TXL alleviates ox-LDL-induced hyperpermeability and provide the basis for further investigations of TXL as regulators of vascular barrier function.

  14. Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients

    PubMed Central

    Mirsepasi-Lauridsen, Hengameh Chloé; Du, Zhengyu; Struve, Carsten; Charbon, Godefroid; Karczewski, Jurgen; Krogfelt, Karen Angeliki; Petersen, Andreas Munk; Wells, Jerry M

    2016-01-01

    Objectives: The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated. Methods: E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls. Results: E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001). Conclusions: UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD. PMID:26938480

  15. [Relationship between tight junction proteins and Helicobacter pylori-associated gastric diseases].

    PubMed

    Li, Wei; Jiang, Mi-Zu

    2014-03-01

    Helicobacter pylori (Hp) infection is an important cause of chronic gastritis and peptic ulcer, but their pathogenesis is unclear. The role of gastric mucosal barrier dysfunction induced by impaired structure and function of tight junction in the pathogenesis of Hp-associated gastric diseases has received considerable attention in recent years. Tight junction is composed of a variety of proteins and molecules, including 3 integral membrane proteins (occludin, claudins, and junctional adhesion molecules) and a cytoplasmic protein (zonula occludens). This paper mainly describes the composition and function of various tight junction proteins, changes in tight junction protein function induced by Hp infection and their relationship with the incidence of gastric diseases, and the significance of enhancing the tight junction protein function in the prevention and treatment of Hp-associated gastric diseases.

  16. Specificity of Interaction between Clostridium perfringens Enterotoxin and Claudin-Family Tight Junction Proteins

    PubMed Central

    Mitchell, Leslie A.; Koval, Michael

    2010-01-01

    Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, forms physical pores in the plasma membrane of intestinal epithelial cells. The ability of CPE to recognize the epithelium is due to the C-terminal binding domain, which binds to a specific motif on the second extracellular loop of tight junction proteins known as claudins. The interaction between claudins and CPE plays a key role in mediating CPE toxicity by facilitating pore formation and by promoting tight junction disassembly. Recently, the ability of CPE to distinguish between specific claudins has been used to develop tools for studying roles for claudins in epithelial barrier function. Moreover, the high affinity of CPE to selected claudins makes CPE a useful platform for targeted drug delivery to tumors expressing these claudins. PMID:22069652

  17. Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

    PubMed

    Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2015-02-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight

  18. Paracellin-1 and the modulation of ion selectivity of tight junctions.

    PubMed

    Hou, Jianghui; Paul, David L; Goodenough, Daniel A

    2005-11-01

    Tight junctions play a key selectivity role in the paracellular conductance of ions. Paracellin-1 is a member of the tight junction claudin protein family and mutations in the paracellin-1 gene cause a human hereditary disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe renal Mg2+ wasting. The mechanism of paracellin-1 function and its role in FHHNC are not known. Here, we report that in LLC-PK1 epithelial cells paracellin-1 modulated the ion selectivity of the tight junction by selectively and significantly increasing the permeability of Na+ (with no effects on Cl-) and generated a high permeability ratio of Na+ to Cl-. Mutagenesis studies identified a locus of amino acids in paracellin-1 critical for this function. Mg2+ flux across cell monolayers showed a far less-pronounced change (compared to monovalent alkali cations) following exogenous protein expression, suggesting that paracellin-1 did not form Mg2+-selective paracellular channels. We hypothesize that in the thick ascending limb of the nephron, paracellin-1 dysfunction, with a concomitant loss of cation selectivity, could contribute to the dissipation of the lumen-positive potential that is the driving force for the reabsorption of Mg2+.

  19. Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-β.

    PubMed

    Schamberger, Andrea C; Mise, Nikica; Jia, Jie; Genoyer, Emmanuelle; Yildirim, Ali Ö; Meiners, Silke; Eickelberg, Oliver

    2014-06-01

    The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults, such as cigarette smoke, environmental particles, or viruses. Although bronchial epithelial integrity is crucial for airway homeostasis, defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure, the major risk factor for COPD, is suggested to impair tight junction integrity; however, detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-β1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically, CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane, as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo, as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-β1 restored ZO-1 and ZO-2 levels. TGF-β1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion, CSE led to the disruption of tight junctions of human bronchial epithelial cells, and TGF-β1 counteracted this CSE-induced effect. Thus, TGF-β1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD.

  20. Enteric Pathogens and Their Toxin-Induced Disruption of the Intestinal Barrier through Alteration of Tight Junctions in Chickens.

    PubMed

    Awad, Wageha A; Hess, Claudia; Hess, Michael

    2017-02-10

    Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing bird's health. The intestinal epithelial barrier serves as the first line of defense between the host and the luminal environment. It consists of a continuous monolayer of intestinal epithelial cells connected by intercellular junctional complexes which shrink the space between adjacent cells. Consequently, free passing of solutes and water via the paracellular pathway is prevented. Tight junctions (TJs) are multi-protein complexes which are crucial for the integrity and function of the epithelial barrier as they not only link cells but also form channels allowing permeation between cells, resulting in epithelial surfaces of different tightness. Tight junction's molecular composition, ultrastructure, and function are regulated differently with regard to physiological and pathological stimuli. Both in vivo and in vitro studies suggest that reduced tight junction integrity greatly results in a condition commonly known as "leaky gut". A loss of barrier integrity allows the translocation of luminal antigens (microbes, toxins) via the mucosa to access the whole body which are normally excluded and subsequently destroys the gut mucosal homeostasis, coinciding with an increased susceptibility to systemic infection, chronic inflammation and malabsorption. There is considerable evidence that the intestinal barrier dysfunction is an important factor contributing to the pathogenicity of some enteric bacteria. It has been shown that some enteric pathogens can induce permeability defects in gut epithelia by altering tight junction proteins, mediated by their toxins. Resolving the strategies that microorganisms use to hijack the functions of tight junctions is important for our understanding of microbial pathogenesis, because some pathogens can

  1. Manufactured Aluminum Oxide Nanoparticles Decrease Expression of Tight Junction Proteins in Brain Vasculature

    PubMed Central

    Yokel, Robert A.; Hennig, Bernhard

    2009-01-01

    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. PMID:18830698

  2. 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.

  3. 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.

  4. Enteric Pathogens and Their Toxin-Induced Disruption of the Intestinal Barrier through Alteration of Tight Junctions in Chickens

    PubMed Central

    Awad, Wageha A.; Hess, Claudia; Hess, Michael

    2017-01-01

    Maintaining a healthy gut environment is a prerequisite for sustainable animal production. The gut plays a key role in the digestion and absorption of nutrients and constitutes an initial organ exposed to external factors influencing bird’s health. The intestinal epithelial barrier serves as the first line of defense between the host and the luminal environment. It consists of a continuous monolayer of intestinal epithelial cells connected by intercellular junctional complexes which shrink the space between adjacent cells. Consequently, free passing of solutes and water via the paracellular pathway is prevented. Tight junctions (TJs) are multi-protein complexes which are crucial for the integrity and function of the epithelial barrier as they not only link cells but also form channels allowing permeation between cells, resulting in epithelial surfaces of different tightness. Tight junction’s molecular composition, ultrastructure, and function are regulated differently with regard to physiological and pathological stimuli. Both in vivo and in vitro studies suggest that reduced tight junction integrity greatly results in a condition commonly known as “leaky gut”. A loss of barrier integrity allows the translocation of luminal antigens (microbes, toxins) via the mucosa to access the whole body which are normally excluded and subsequently destroys the gut mucosal homeostasis, coinciding with an increased susceptibility to systemic infection, chronic inflammation and malabsorption. There is considerable evidence that the intestinal barrier dysfunction is an important factor contributing to the pathogenicity of some enteric bacteria. It has been shown that some enteric pathogens can induce permeability defects in gut epithelia by altering tight junction proteins, mediated by their toxins. Resolving the strategies that microorganisms use to hijack the functions of tight junctions is important for our understanding of microbial pathogenesis, because some pathogens

  5. Novel Effects of Azithromycin on Tight Junction Proteins in Human Airway Epithelia

    PubMed Central

    Asgrimsson, Valthor; Gudjonsson, Thorarinn; Gudmundsson, Gudmundur Hrafn; Baldursson, Olafur

    2006-01-01

    The macrolide antibiotic azithromycin improves lung function and prognosis among patients with cystic fibrosis or diffuse panbronchiolitis, independently of bacterial eradication. Anti-inflammatory effects have been implicated, but data from in vivo studies are scarce, and the link between abnormal electrolyte content in airway surface liquid and bronchial infections remains uncertain. In the present study, we treated human airway epithelia on filter supports with azithromycin and monitored transepithelial electrical resistance. We found that azithromycin increased transepithelial electrical resistance of airway epithelia in a dose-dependent manner. Immunocytochemistry and Western blotting showed that addition of azithromycin changed the locations of proteins in cell cultures and induced processing of the tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A. These effects were reversible, and no effect was seen when cells were treated with penicillin or erythromycin. The data indicate that azithromycin increases the transepithelial electrical resistance of human airway epithelia by changing the processing of tight junction proteins. The results are novel and may help explain the beneficial effects of azithromycin in patients with cystic fibrosis, diffuse panbronchiolitis, and community-acquired pneumonia. PMID:16641453

  6. Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells.

    PubMed

    Alvarez, Carina-Shianya; Badia, Josefa; Bosch, Manel; Giménez, Rosa; Baldomà, Laura

    2016-01-01

    The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional

  7. Outer Membrane Vesicles and Soluble Factors Released by Probiotic Escherichia coli Nissle 1917 and Commensal ECOR63 Enhance Barrier Function by Regulating Expression of Tight Junction Proteins in Intestinal Epithelial Cells

    PubMed Central

    Alvarez, Carina-Shianya; Badia, Josefa; Bosch, Manel; Giménez, Rosa; Baldomà, Laura

    2016-01-01

    The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional

  8. A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

    PubMed Central

    Van Itallie, Christina M.; Tietgens, Amber Jean; Krystofiak, Evan; Kachar, Bechara; Anderson, James M.

    2015-01-01

    Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin. PMID:26063734

  9. Transcription factor AP-2γ is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis

    PubMed Central

    Choi, Inchul; Carey, Timothy S.; Wilson, Catherine A.; Knott, Jason G.

    2012-01-01

    The trophectoderm epithelium is the first differentiated cell layer to arise during mammalian development. Blastocyst formation requires the proper expression and localization of tight junction, polarity, ion gradient and H2O channel proteins in the outer cell membranes. However, the underlying transcriptional mechanisms that control their expression are largely unknown. Here, we report that transcription factor AP-2γ (Tcfap2c) is a core regulator of blastocyst formation in mice. Bioinformatics, chromatin immunoprecipitation and transcriptional analysis revealed that Tcfap2c binds and regulates a diverse group of genes expressed during blastocyst formation. RNA interference experiments demonstrated that Tcfap2c regulates genes important for tight junctions, cell polarity and fluid accumulation. Functional and ultrastructural studies revealed that Tcfap2c is necessary for tight junction assembly and paracellular sealing in trophectoderm epithelium. Aggregation of control eight-cell embryos with Tcfap2c knockdown embryos rescued blastocyst formation via direct contribution to the trophectoderm epithelium. Finally, we found that Tcfap2c promotes cellular proliferation via direct repression of p21 transcription during the morula-to-blastocyst transition. We propose a model in which Tcfap2c acts in a hierarchy to facilitate blastocyst formation through transcriptional regulation of core genes involved in tight junction assembly, fluid accumulation and cellular proliferation. PMID:23136388

  10. PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities

    PubMed Central

    Huang, Wen; Eum, Sung Yong; András, Ibolya E; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    The blood-brain barrier (BBB) plays an important role in HIV trafficking into the brain and the development of the central nervous system complications in HIV infection. Tight junctions are the main structural and functional elements that regulate the BBB integrity. Exposure of human brain microvascular endothelial cells (hCMEC/D3 cell line) to HIV-infected monocytes resulted in decreased expression of tight junction proteins, such as junctional adhesion molecule-A (JAM)-A, occludin, and zonula occludens (ZO)-1. Control experiments involved exposure to uninfected monocytes. Alterations of tight junction protein expression were associated with increased endothelial permeability and elevated transendothelial migration of HIV-infected monocytes across an in vitro model of the BBB. Notably, overexpression of the peroxisome proliferator-activated receptor (PPAR)α or PPARγ attenuated HIV-mediated dysregulation of tight junction proteins. With the use of exogenous PPARγ agonists and silencing of PPARα or PPARγ, these protective effects were connected to down-regulation of matrix metalloproteinase (MMP) and proteasome activities. Indeed, the HIV-induced decrease in the expression of JAM-A and occludin was restored by inhibition of MMP activity. Moreover, both MMP and proteasome inhibitors attenuated HIV-mediated altered expression of ZO-1. The present data indicate that down-regulation of MMP and proteasome activities constitutes a novel mechanism of PPAR-induced protections against HIV-induced disruption of brain endothelial cells.—Huang, W., Eum, S. Y., András, I. E., Hennig, B., Toborek, M. PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. PMID:19141539

  11. Digestion of epithelial tight junction proteins by the commensal Clostridium perfringens.

    PubMed

    Pruteanu, Mihaela; Shanahan, Fergus

    2013-11-15

    The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens (C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.

  12. Keratin 76 is required for tight junction function and maintenance of the skin barrier.

    PubMed

    DiTommaso, Tia; Cottle, Denny L; Pearson, Helen B; Schlüter, Holger; Kaur, Pritinder; Humbert, Patrick O; Smyth, Ian M

    2014-10-01

    Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.

  13. Keratin 76 Is Required for Tight Junction Function and Maintenance of the Skin Barrier

    PubMed Central

    DiTommaso, Tia; Cottle, Denny L.; Pearson, Helen B.; Schlüter, Holger; Kaur, Pritinder; Humbert, Patrick O.; Smyth, Ian M.

    2014-01-01

    Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases. PMID:25340345

  14. HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

    PubMed Central

    Saeedi, Bejan J.; Kao, Daniel J.; Kitzenberg, David A.; Dobrinskikh, Evgenia; Schwisow, Kayla D.; Masterson, Joanne C.; Kendrick, Agnieszka A.; Kelly, Caleb J.; Bayless, Amanda J.; Kominsky, Douglas J.; Campbell, Eric L.; Kuhn, Kristine A.; Furuta, Glenn T.; Colgan, Sean P.; Glover, Louise E.

    2015-01-01

    Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA–mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis. PMID:25904334

  15. The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding

    PubMed Central

    Marchiando, Amanda M.; Shen, Le; Graham, W. Vallen; Edelblum, Karen L.; Duckworth, Carrie A.; Guan, Yanfang; Montrose, Marshall H.; Turner, Jerrold R.; Watson, Alastair J.M.

    2011-01-01

    BACKGROUND & AIMS Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance, as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. METHODS We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein1–ZO-1. After injection of high doses of TNF (7.5µg, i.p.), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. RESULTS Changes detected included redistribution of the tight junction proteins ZO-1 and occluding to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. CONCLUSIONS Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function. PMID:21237166

  16. 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

  17. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-08-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events.

  18. Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters

    PubMed Central

    Wang, Xinyi; Wang, Na; Yuan, Lan; Li, Na; Wang, Junxia; Yang, Xiaoda

    2016-01-01

    Tight junctions play a key role in restricting or regulating passage of liquids, ions and large solutes through various biological barriers by the paracellular route. Changes in paracellular permeation indicate alteration of the tight junction. However, it is very difficult to obtain the structural change information by measuring paracellular flux based on transepithelial electrical resistance or using fluorescein-labeled dextrans. Here we show that the BSA and GSH stabilized gold nanoclusters exhibit marginal cytotoxicity and pass through the MDCK monolayer exclusively through the paracellular pathway. We propose a double fluorescence probe strategy, the combination of a proven paracellular indicator (europium complex) with fluorescent gold nanoclusters. We calculate changes of structural parameters in tight junctions based on determination of the diffusion coefficients of the probes. Two different types of tight junction openers are used to validate our strategy. Results show that EDTA disrupts tight junction structures and induces large and smooth paracellular pore paths with an average radius of 17 nm, but vanadyl complexes induce paths with the radius of 6 nm. The work suggests that the double fluorescence probe strategy is a useful and convenient approach for in vitro investigation of tight junction structural alternations caused by pharmacological or pathological events. PMID:27574102

  19. Structure of the tight junctions of the human eccrine sweat gland.

    PubMed

    Briggman, J V; Bank, H L; Bigelow, J B; Graves, J S; Spicer, S S

    1981-12-01

    The human eccrine sweat gland contains two anatomically and functionally discrete segments: the secretory coil, which produces an isotonic or slightly hypertonic precursor fluid, and the coiled duct, which reabsorbs Na+ and Cl- to yield a hypotonic sweat. We examined the freeze-fracture morphology of tight junctions from isolated secretory coil and coiled duct segments to assess indirectly the contribution of paracellular ion transport in secretion and resorption in the sweat gland. In the secretory coil, tight junctions of the intercellular canaliculus and main lumen consisted of approximately 9 and 6, closely spaced, parallel or anastomosing elements, respectively. Tight junctions of the coiled duct were similar in appearance to those at the main lumen of the secretory coil. In both the secretory coil and coiled duct, and average of 2 to 3, widely spaced junctional elements were usually observed basolateral to the closely spaced junctional elements in the region corresponding to the location of the zonula adherens in Epon sections. The complexity of the tight junctions of the secretory coil exceeded what we expected for an epithelium secreting an isosmotic fluid. The elaborate tight junctions of the coiled duct support other evidence for an intermediate to high transepithelial resistance.

  20. Dot junction solar cells

    NASA Technical Reports Server (NTRS)

    Daud, T.; Crotty, G. T.

    1986-01-01

    A design of solar cells with reduced junction area on the cell surface is investigated for reduction of saturation current and increase in open-circuit voltage. Equidiameter dot junctions distributed across the surface of the cell offer an efficient alternative, with variations in dot diameter and in the spacing between dots giving the required variations in the ratio of junction area to total surface area. A simplified analysis for short-circuit current and other cell parameters, which enables cell design optimization, is presented. Experimental solar-cell performance results, as functions of different area ratios, are presented and compared with the model. It is shown that saturation current reduction is possible for achieving efficiencies as high as 18 percent in flat-plate terrestrial applications.

  1. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

    PubMed

    Sonoda, N; Furuse, M; Sasaki, H; Yonemura, S; Katahira, J; Horiguchi, Y; Tsukita, S

    1999-10-04

    Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

  2. Quantum junction solar cells.

    PubMed

    Tang, Jiang; Liu, Huan; Zhitomirsky, David; Hoogland, Sjoerd; Wang, Xihua; Furukawa, Melissa; Levina, Larissa; Sargent, Edward H

    2012-09-12

    Colloidal quantum dot solids combine convenient solution-processing with quantum size effect tuning, offering avenues to high-efficiency multijunction cells based on a single materials synthesis and processing platform. The highest-performing colloidal quantum dot rectifying devices reported to date have relied on a junction between a quantum-tuned absorber and a bulk material (e.g., TiO(2)); however, quantum tuning of the absorber then requires complete redesign of the bulk acceptor, compromising the benefits of facile quantum tuning. Here we report rectifying junctions constructed entirely using inherently band-aligned quantum-tuned materials. Realizing these quantum junction diodes relied upon the creation of an n-type quantum dot solid having a clean bandgap. We combine stable, chemically compatible, high-performance n-type and p-type materials to create the first quantum junction solar cells. We present a family of photovoltaic devices having widely tuned bandgaps of 0.6-1.6 eV that excel where conventional quantum-to-bulk devices fail to perform. Devices having optimal single-junction bandgaps exhibit certified AM1.5 solar power conversion efficiencies of 5.4%. Control over doping in quantum solids, and the successful integration of these materials to form stable quantum junctions, offers a powerful new degree of freedom to colloidal quantum dot optoelectronics.

  3. 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

  4. Dehydroepiandrosterone Sulfate Stimulates Expression of Blood-Testis-Barrier Proteins Claudin-3 and -5 and Tight Junction Formation via a Gnα11-Coupled Receptor in Sertoli Cells

    PubMed Central

    Papadopoulos, Dimitrios; Dietze, Raimund; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2016-01-01

    Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the

  5. The Tight Junction Proteins Claudin-1, -6, and -9 Are Entry Cofactors for Hepatitis C Virus▿

    PubMed Central

    Meertens, Laurent; Bertaux, Claire; Cukierman, Lisa; Cormier, Emmanuel; Lavillette, Dimitri; Cosset, François-Loïc; Dragic, Tatjana

    2008-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81+ cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis. PMID:18234789

  6. Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions

    PubMed Central

    Xiao, Chang; Ogle, Sally A.; Schumacher, Michael A.; Schilling, Neal; Tokhunts, Robert A.; Orr-Asman, Melissa A.; Miller, Marian L.; Robbins, David J.; Hollande, Frederic

    2010-01-01

    In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5Ski) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/ShhKO) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5Ski cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/ShhKO mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1. PMID:20847300

  7. 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

  8. Effect of four probiotic strains and Escherichia coli O157:H7 on tight junction integrity and cyclo-oxygenase expression.

    PubMed

    Putaala, Heli; Salusjärvi, Tuomas; Nordström, Malin; Saarinen, Markku; Ouwehand, Arthur C; Bech Hansen, Egon; Rautonen, Nina

    2008-01-01

    Controversy exists as to whether contact between a probiotic bacterial cell and an epithelial cell in the gut is needed to confer beneficial effects of probiotics, or whether metabolites from probiotics are sufficient to cause this effect. To address this question, Caco-2 cells were treated with cell-free supernatants of four probiotics, Bifidobacterium lactis 420, Bifidobacterium lactis HN019, Lactobacillus acidophilus NCFM, Lactobacillus salivarius Ls-33, and by a cell-free supernatant of a pathogenic bacteria, Escherichia coli O157:H7 (EHEC). Tight junction integrity as well as expression of cyclo-oxygenases, which are prostaglandin-producing enzymes, were measured. Probiotic-specific as well as EHEC-specific effects on tight junction integrity and cyclo-oxygenase expression were evident, indicating that live bacterial cells were not necessary for the manifestation of the effects. B. lactis 420 cell-free supernatant increased tight junction integrity, while EHEC cell-free supernatant induced damage on tight junctions. In general, EHEC and probiotics had opposite effects upon cyclo-oxygenase expression. Furthermore, B. lactis 420 cell-free supernatant protected the tight junctions from EHEC-induced damage when administered prior to the cell-free supernatant of EHEC. These results indicate that probiotics produce bioactive metabolites, suggesting that consumption of specific probiotic bacteria might be beneficial in protecting intestinal epithelial cells from the deleterious effects of pathogenic bacteria.

  9. 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

    2017-02-10

    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.

  10. Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability.

    PubMed Central

    Cuzzocrea, S.; Mazzon, E.; De Sarro, A.; Caputi, A. P.

    2000-01-01

    BACKGROUND: Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. MATERIALS AND METHODS: In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. RESULTS: Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with

  11. Differential expression of claudin tight junction proteins in the human cortical nephron

    PubMed Central

    Kirk, Adam; Campbell, Sara; Bass, Paul; Mason, Juan; Collins, Jane

    2010-01-01

    Background. In renal tubules, paracellular permeability is tightly controlled to facilitate solute absorption and urinary concentration and is regulated by tight junctions, which incorporate claudin proteins. There is very limited information confirming the localization of these proteins in the human renal cortex. Most data is inferred from mouse, bovine and rabbit studies and differences exist between mouse and other species. Methods. A survey of claudin staining was performed on human kidney cortex embedded in glycolmethacrylate resin to enhance tissue morphology and facilitate the cutting of 2 µm serial sections. Results. Claudin-2, -10 and -11 antibodies labelled renal tubular epithelial cells, correlating with Lotus tetragonolobus and N-cadherin positive proximal tubules. Claudin-3, -10, -11 and -16 antibodies strongly stained a population of tubules that were positive for Tamm Horsfall protein on adjacent sections, confirming expression in the thick ascending limb of the Loop of Henle. Claudin-3, -4 and -8 antibodies reacted with tubules that correlated with the distal nephron markers, E-cadherin, epithelial membrane antigen and Dolichos biflorus and claudin-3, -4, -7 and -8 with the distal tubule marker, calbindin, and the collecting duct marker, aquaporin-2. Claudin-14 was localized in distal convoluted tubules, correlating positively with calbindin but negatively with aquaporin-2, whereas claudin-1 staining was identified in the parietal epithelium of Bowman's capsule, distal convoluted tubule and collecting duct. Cellular and tight junction localization of claudin staining in renal tubules was heterogeneous and is discussed. Conclusions. Complex variation in the expression of human claudins likely determines paracellular permeability in the kidney. Altered claudin expression may influence pathologies involving abnormalities of absorption. PMID:20124215

  12. The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

    PubMed Central

    Yu, Changsong; Jia, Gang; Deng, Qiuhong; Zhao, Hua; Chen, Xiaoling; Liu, Guangmang; Wang, Kangning

    2016-01-01

    Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway. PMID:26954146

  13. Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer

    PubMed Central

    Shimada, Hiroshi; Abe, Shyuetsu; Kohno, Takayuki; Satohisa, Seiro; Konno, Takumi; Takahashi, Syunta; Hatakeyama, Tsubasa; Arimoto, Chihiro; Kakuki, Takuya; Kaneko, Yakuto; Takano, Ken-ichi; Saito, Tsuyoshi; Kojima, Takashi

    2017-01-01

    Lipolysis-stimulated lipoprotein receptor (LSR) is a unique molecule of tricellular contacts of normal and cancer cells. We investigated how the loss of LSR induced cell migration, invasion and proliferation in endometrial cancer cell line Sawano. mRNAs of amphiregulin (AREG) and TEA domain family member 1 (TEAD1) were markedly upregulated by siRNA-LSR. In endometrial cancer tissues, downregulation of LSR and upregulation of AREG were observed together with malignancy, and Yes-associated protein (YAP) was present in the nuclei. siRNA-AREG prevented the cell migration and invasion induced by siRNA-LSR, whereas treatment with AREG induced cell migration and invasion. LSR was colocalized with TRIC, angiomotin (AMOT), Merlin and phosphorylated YAP (pYAP). siRNA-LSR increased expression of pYAP and decreased that of AMOT and Merlin. siRNA-YAP prevented expression of the mRNAs of AREG and TEAD1, and the cell migration and invasion induced by siRNA-LSR. Treatment with dobutamine and 2-deoxy-D-glucose and glucose starvation induced the pYAP expression and prevented the cell migration and invasion induced by siRNA-LSR. siRNA-AMOT decreased the Merlin expression and prevented the cell migration and invasion induced by siRNA-LSR. The loss of LSR promoted cell invasion and migration via upregulation of TEAD1/AREG dependent on YAP/pYAP and AMOT/Merlin in human endometrial cancer cells. PMID:28071680

  14. The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction.

    PubMed

    Ronaghan, Natalie J; Shang, Judie; Iablokov, Vadim; Zaheer, Raza; Colarusso, Pina; Dion, Sébastien; Désilets, Antoine; Leduc, Richard; Turner, Jerrold R; MacNaughton, Wallace K

    2016-09-01

    Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

  15. 17β-Estradiol Ameliorates Tight Junction Disruption via Repression of MMP Transcription.

    PubMed

    Na, Wonho; Lee, Jee Youn; Kim, Won-Sun; Yune, Tae Young; Ju, Bong-Gun

    2015-09-01

    The blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) formed by capillary endothelial cells provides a physical wall between the central nervous system (CNS) and circulating blood with highly selective permeability. BBB/BSCB disruption by activation of matrix metalloproteinases (MMPs) has been shown to result in further neurological damage after CNS injury. Recently it has been discovered that estrogen attenuates BBB/BSCB disruption in in vitro and in vivo models. However, the molecular mechanism underlying the estrogen-mediated attenuation of BBB/BSCB disruption has not been elucidated fully. In the present study, we found that 17β-estradiol (E2) suppresses nuclear factor-κB-dependent MMP-1b, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 gene activation in microvessel endothelial bEnd.3 cells subjected to oxygen and glucose deprivation/reperfusion injury. E2 induced the recruitment of ERα and nuclear receptor corepressor to the nuclear factor-κB binding site on the MMPs' gene promoters. Consistently, ER antagonist ICI 182.780 showed opposite effects of E2. We further found that E2 attenuates tight junction disruption through the decreased degradation of tight junction proteins in bEnd.3 cells subjected to oxygen and glucose deprivation-reperfusion injury. In addition, E2 suppressed the up-regulation of MMP expression, leading to a decreased BSCB disruption in the injured spinal cord. In conclusion, we discovered the molecular mechanism underlying the protective role of estrogenin BBB/BSCB disruption using an in vitro and in vivo model. Our study suggests that estrogens may provide a potential therapeutic intervention for preserving BBB/BSCB integrity after CNS injury.

  16. 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

  17. Alteration of Tight Junction Proteins Is an Early Event in Psoriasis

    PubMed Central

    Kirschner, Nina; Poetzl, Claudia; von den Driesch, Peter; Wladykowski, Ewa; Moll, Ingrid; Behne, Martin J.; Brandner, Johanna M.

    2009-01-01

    Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis. Here we show that localization of TJ proteins is already altered in early-stage psoriasis. Occludin, ZO-1, and claudin-4 are found in more layers than in normal epidermis, and claudin-1 and -7 are down-regulated in the basal and in the uppermost layers. In plaque-type psoriasis, the staining patterns of occludin and ZO-1 do not change, whereas the claudins are further down-regulated. Near transmigrating granulocytes, all TJ proteins except for junctional adhesion molecule-A are down-regulated. Treatment of cultured keratinocytes with interleukin-1β and tumor necrosis factor-α, which are present at elevated levels in psoriatic skin, results in an increase of transepithelial resistance at early time points and a decrease at later time points. Injection of interleukin-1β into an ex vivo skin model leads to an up-regulation of occludin and ZO-1, resembling TJ protein alteration in early psoriasis. Our results show for the first time that alteration of TJ proteins is an early event in psoriasis and is not the consequence of the more profound changes found in plaque-type psoriasis. Our data indicate that cytokines are involved in alterations of TJ proteins observed in psoriasis. PMID:19661441

  18. Irinotecan disrupts tight junction proteins within the gut : implications for chemotherapy-induced gut toxicity.

    PubMed

    Wardill, Hannah R; Bowen, Joanne M; Al-Dasooqi, Noor; Sultani, Masooma; Bateman, Emma; Stansborough, Romany; Shirren, Joseph; Gibson, Rachel J

    2014-02-01

    Chemotherapy for cancer causes significant gut toxicity, leading to severe clinical manifestations and an increased economic burden. Despite much research, many of the underlying mechanisms remain poorly understood hindering effective treatment options. Recently there has been renewed interest in the role tight junctions play in the pathogenesis of chemotherapy-induced gut toxicity. To delineate the underlying mechanisms of chemotherapy-induced gut toxicity, this study aimed to quantify the molecular changes in key tight junction proteins, ZO-1, claudin-1, and occludin, using a well-established preclinical model of gut toxicity. Female tumor-bearing dark agouti rats received irinotecan or vehicle control and were assessed for validated parameters of gut toxicity including diarrhea and weight loss. Rats were killed at 6, 24, 48, 72, 96, and 120 h post-chemotherapy. Tight junction protein and mRNA expression in the small and large intestines were assessed using semi-quantitative immunohistochemistry and RT-PCR. Significant changes in protein expression of tight junction proteins were seen in both the jejunum and colon, correlating with key histological changes and clinical features. mRNA levels of claudin-1 were significantly decreased early after irinotecan in the small and large intestines. ZO-1 and occludin mRNA levels remained stable across the time-course of gut toxicity. Findings strongly suggest irinotecan causes tight junction defects which lead to mucosal barrier dysfunction and the development of diarrhea. Detailed research is now warranted to investigate posttranslational regulation of tight junction proteins to delineate the underlying pathophysiology of gut toxicity and identify future therapeutic targets.

  19. Tight Junctions: A Barrier to the Initiation and Progression of Breast Cancer?

    PubMed Central

    Brennan, Kieran; Offiah, Gozie; McSherry, Elaine A.; Hopkins, Ann M.

    2010-01-01

    Breast cancer is a complex and heterogeneous disease that arises from epithelial cells lining the breast ducts and lobules. Correct adhesion between adjacent epithelial cells is important in determining the normal structure and function of epithelial tissues, and there is accumulating evidence that dysregulated cell-cell adhesion is associated with many cancers. This review will focus on one cell-cell adhesion complex, the tight junction (TJ), and summarize recent evidence that TJs may participate in breast cancer development or progression. We will first outline the protein composition of TJs and discuss the functions of the TJ complex. Secondly we will examine how alterations in these functions might facilitate breast cancer initiation or progression; by focussing on the regulatory influence of TJs on cell polarity, cell fate and cell migration. Finally we will outline how pharmacological targeting of TJ proteins may be useful in limiting breast cancer progression. Overall we hope to illustrate that the relationship between TJ alterations and breast cancer is a complex one; but that this area offers promise in uncovering fundamental mechanisms linked to breast cancer progression. PMID:19920867

  20. Cellular mechanisms of mainstream cigarette smoke-induced lung epithelial tight junction permeability changes in vitro.

    PubMed

    Olivera, Dorian S; Boggs, Susan E; Beenhouwer, Chris; Aden, James; Knall, Cindy

    2007-01-01

    Mainstream cigarette smoke increases the permeability of human airways; however, the mechanism for this increased permeability is poorly defined. Tight junctions between adjacent epithelial cells constitute the physiological barrier to fluid and macromolecules in epithelium. These structures are highly regulated by phosphorylation and their association with the cytoskeleton. The goal of these studies was to identify the signal transduction pathways that regulate smoke-induced permeability. Using a physiologically relevant air-liquid interface exposure system, electrically tight monolayers of the human bronchial epithelial cell-line Calu-3 were exposed to fresh, whole mainstream cigarette smoke. This exposure results in a regulated, dose-dependent loss of epithelial barrier function in the lung epithelial monolayers. With cigarette smoke exposure, transepithelial electrical resistance (TER) is decreased and albumin flux is increased, indicating a loss in barrier function to ions and macromolecules, respectively; however, both largely recover in 30 min. Smoke-induced losses of macromolecular barrier function are the result of multicellular junctional reorganization, resulting in increased leak volume rather than leak frequency. Inhibiting Rho kinase (ROCK) significantly reduces the smoke-induced permeability to both ions and macromolecules, while inhibiting protein tyrosine kinases (PTK) only reduces smoke-induced macromolecular permeability. Interestingly, inhibiting myosin light chain kinase (MLCK) exacerbates smoke-induced permeability, indicating that MLCK and ROCK have opposing regulatory roles. Our results demonstrate that the smoke-induced loss of epithelial barrier function in human bronchial epithelium is a regulated process rather than a cytotoxic response. Additionally, our results indicate that activation of PTK and ROCK and inactivation of MLCK contribute to the increased airway permeability caused by mainstream cigarette smoke.

  1. Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas

    PubMed Central

    1979-01-01

    The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P- faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E- face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P- face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results. PMID:457764

  2. Organization and formation of the tight junction system in human epidermis and cultured keratinocytes.

    PubMed

    Brandner, Johanna M; Kief, Sabine; Grund, Christine; Rendl, Michael; Houdek, Pia; Kuhn, Caecilia; Tschachler, Erwin; Franke, Werner W; Moll, Ingrid

    2002-05-01

    Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are

  3. 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.

  4. ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

    PubMed Central

    Kwon, Jeongwoo

    2016-01-01

    The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins. PMID:27077008

  5. Delivery of Berberine Using Chitosan/Fucoidan-Taurine Conjugate Nanoparticles for Treatment of Defective Intestinal Epithelial Tight Junction Barrier

    PubMed Central

    Wu, Shao-Jung; Don, Trong-Ming; Lin, Cheng-Wei; Mi, Fwu-Long

    2014-01-01

    Bacterial-derived lipopolysaccharides (LPS) can cause defective intestinal barrier function and play an important role in the development of inflammatory bowel disease. In this study, a nanocarrier based on chitosan and fucoidan was developed for oral delivery of berberine (Ber). A sulfonated fucoidan, fucoidan-taurine (FD-Tau) conjugate, was synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy. The FD-Tau conjugate was self-assembled with berberine and chitosan (CS) to form Ber-loaded CS/FD-Tau complex nanoparticles with high drug loading efficiency. Berberine release from the nanoparticles had fast release in simulated intestinal fluid (SIF, pH 7.4), while the release was slow in simulated gastric fluid (SGF, pH 2.0). The effect of the berberine-loaded nanoparticles in protecting intestinal tight-junction barrier function against nitric oxide and inflammatory cytokines released from LPS-stimulated macrophage was evaluated by determining the transepithelial electrical resistance (TEER) and paracellular permeability of a model macromolecule fluorescein isothiocyanate-dextran (FITC-dextran) in a Caco-2 cells/RAW264.7 cells co-culture system. Inhibition of redistribution of tight junction ZO-1 protein by the nanoparticles was visualized using confocal laser scanning microscopy (CLSM). The results suggest that the nanoparticles may be useful for local delivery of berberine to ameliorate LPS-induced intestinal epithelia tight junction disruption, and that the released berberine can restore barrier function in inflammatory and injured intestinal epithelial. PMID:25421323

  6. Interleukin-8 Regulates Endothelial Permeability by Down-regulation of Tight Junction but not Dependent on Integrins Induced Focal Adhesions

    PubMed Central

    Yu, Hongchi; Huang, Xianliang; Ma, Yunlong; Gao, Min; Wang, Ou; Gao, Ting; Shen, Yang; Liu, Xiaoheng

    2013-01-01

    Interleukin-8 (IL-8) is a common inflammatory factor, which involves in various non-specific pathological processes of inflammation. It has been found that increased endothelial permeability accompanied with high expression of IL-8 at site of injured endothelium and atherosclerotic plaque at early stages, suggesting that IL-8 participated in regulating endothelial permeability in the developing processes of vascular disease. The purpose of this study is to investigate the regulation effects of IL-8 on the vascular endothelial permeability, and the mRNA and protein expression of tight junction components (i.e., ZO-1, Claudin-5 and Occludin). Endothelial cells were stimulated by IL-8 with the dose of 50, 100 and 200 ng/mL, and duration of 2, 4, 6, 8h, respectively. The mRNA and protein expression level of tight junction components with IL-8 under different concentration and duration was examined by RT-PCR and Western blot, respectively. Meanwhile, the integrins induced focal adhesions event with IL-8 stimulation was also investigated. The results showed that IL-8 regulated the permeability of endothelium by down-regulation of tight junction in a dose- and time-dependence manner, but was not by integrins induced focal adhesions. This finding reveals the molecular mechanism in the increase of endothelial cell permeability induced by IL-8, which is expected to provide a new idea as a therapeutic target in vascular diseases. PMID:24155670

  7. Inactivation of hepatocyte nuclear factor-4α mediates alcohol-induced downregulation of intestinal tight junction proteins

    PubMed Central

    Zhong, Wei; Zhao, Yantao; McClain, Craig J.; Kang, Y. James

    2010-01-01

    Chronic alcohol exposure has been shown to increase the gut permeability in the distal intestine, in part, through induction of zinc deficiency. The present study evaluated the molecular mechanisms whereby zinc deficiency mediates alcohol-induced intestinal barrier dysfunction. Examination of zinc finger transcription factors in the gastrointestinal tract of mice revealed a prominent distribution of hepatocyte nuclear factor-4α (HNF-4α). HNF-4α exclusively localizes in the epithelial nuclei and exhibited an increased abundance in mRNA and protein levels in the distal intestine. Chronic alcohol exposure to mice repressed the HNF-4α gene expression in the ileum and reduced the protein level and DNA binding activity of HNF-4α in all of the intestinal segments with the most remarkable changes in the ileum. Chronic alcohol exposure also decreased the mRNA levels of tight junction proteins, particularly in the ileum. Caco-2 cell culture studies were conducted to determine the role of HNF-4α in regulation of the epithelial tight junction and barrier function. Knockdown of HNF-4α in Caco-2 cells decreased the mRNA and protein levels of tight junction proteins in association with disruption of the epithelial barrier. Alcohol treatment inactivated HNF-4α, which was prevented by N-acetyl-cysteine or zinc. The link between zinc and HNF-4α function was confirmed by zinc deprivation, which inhibited HNF-4α DNA binding activity. These results indicate that inactivation of HNF-4α due to oxidative stress and zinc deficiency is likely a novel mechanism contributing to the deleterious effects of alcohol on the tight junctions and the intestinal barrier function. PMID:20576917

  8. Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

    PubMed Central

    Wang, Hui; Zhao, Jun-Xing; Hu, Nan; Ren, Jun; Du, Min; Zhu, Mei-Jun

    2012-01-01

    AIM: To investigate the effect of side-stream smoking on gut microflora composition, intestinal inflammation and expression of tight junction proteins. METHODS: C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks. Cecal contents were collected for microbial composition analysis. Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins. RESULTS: Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria, Clostridium but decreased Fermicutes (Lactoccoci and Ruminococcus), Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice. Meanwhile, side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα, accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6. The contents of tight junction proteins, claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking. In addition, side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling, while inhibiting AMP-activated protein kinase in the large intestine. CONCLUSION: Side-stream smoking altered gut microflora composition and reduced the inflammatory response, which was associated with increased expression of tight junction proteins. PMID:22611310

  9. Sealing the live part of the skin: the integrated meshwork of desmosomes, tight junctions and curvilinear ridge structures in the cells of the uppermost granular layer of the human epidermis.

    PubMed

    Schlüter, Holger; Wepf, Roger; Moll, Ingrid; Franke, Werner W

    2004-12-01

    In the literature the question of whether a system structurally and functionally related to the barrier function of the tight junctions (TJs) of polarized epithelia exists in the epidermis has been and still is controversially discussed. We have systematically addressed this question in a study of the granular layer of fetal and adult human epidermis, combining different light and electron microscopic methods. We show that the lateral membranes of the cells of the stratum granulosum are connected by an extended subapical complex system integrating desmosomes and TJ structures identified as sites of close membrane-membrane contact and as regions of membrane-to-membrane apposition that in immunoelectron microscopy are positive for TJ marker proteins, notably occludin, indicative of an extended, probably continuous TJ barrier. In addition, we have noted in freeze-fractures of the apical membrane attaching this layer to the basalmost membrane of the stratum corneum an extended system integrating desmosomes with intramembraneous ridge configurations that appear as strands, circles, lariats or complex meshworks showing numerous continuities with the desmosomes. In some regions this system interconnecting desmosomes with curvilinear ridge structures occupies the major part of the plasma membrane. The molecular organizations and possible functional contributions of both structural systems positioned at the border between the living portion of the epidermis and the corneal layer are discussed, in particular in relation to the formation of a stable association between the two layers and of a barrier to the paracellular flow of molecules and particles. It is also discussed whether similar structures occur in other keratinizing stratified squamous epithelia, in squamous metaplasias and in tumors derived from such tissues.

  10. Effects of fumaric acid esters on blood-brain barrier tight junction proteins.

    PubMed

    Bénardais, Karelle; Pul, Refik; Singh, Vikramjeet; Skripuletz, Thomas; Lee, De-Hyung; Linker, Ralf A; Gudi, Viktoria; Stangel, Martin

    2013-10-25

    The blood-brain barrier (BBB) is composed of a network of tight junctions (TJ) which interconnect cerebral endothelial cells (EC). Alterations in the TJ proteins are common in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS). Modulation of the BBB could thus represent a therapeutic mechanism. One pathway to modulate BBB integrity could be the induction of nuclear-factor (erythroid derived 2) related factor-2 (Nrf2) mediated oxidative stress responses which are targeted by fumaric acid esters (FAE). Here we analyze effects of FAE on the expression of TJ proteins in the human cerebral endothelial cell line hCMEC/D3 and experimental autoimmune encephalomyelitis (EAE). We show that dimethylfumarate (DMF) and its primary metabolite monomethylfumarate (MMF) induce the expression of the Nrf2/NQO1 pathway in endothelial cells. Neither MMF nor DMF had a consistent modulatory effect on the expression of TJ molecules in hCMEC/D3 cells. Tumor necrosis factor (TNFα)-induced downregulation of TJ proteins was at least partially reversed by treatment with FAE. However, DMF had no effect on claudin-5 expression in EAE, despite its effect on the clinical score and infiltration of immune cells. These data suggest that the modulation of the BBB is not a major mechanism of action of FAE in inflammatory demyelinating diseases of the CNS.

  11. Endothelial STAT3 Activation Increases Vascular Leakage Through Downregulating Tight Junction Proteins: Implications for Diabetic Retinopathy.

    PubMed

    Yun, Jang-Hyuk; Park, Sung Wook; Kim, Kyung-Jin; Bae, Jong-Sup; Lee, Eun Hui; Paek, Sun Ha; Kim, Seung U; Ye, Sangkyu; Kim, Jeong-Hun; Cho, Chung-Hyun

    2017-05-01

    Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 232: 1123-1134, 2017. © 2016 Wiley Periodicals, Inc.

  12. Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes.

    PubMed

    Milatz, Susanne; Krug, Susanne M; Rosenthal, Rita; Günzel, Dorothee; Müller, Dominik; Schulzke, Jörg-Dieter; Amasheh, Salah; Fromm, Michael

    2010-11-01

    The paracellular barrier of epithelia and endothelia is established by several tight junction proteins including claudin-3. Although claudin-3 is present in many epithelia including skin, lung, kidney, and intestine and in endothelia, its function is unresolved as yet. We therefore characterized claudin-3 by stable transfection of MDCK II kidney tubule cells with human claudin-3 cDNA. Two clone systems were analyzed, exhibiting high or low claudin-2 expression, respectively. Expression of other claudins was unchanged. Ultrastructurally, tight junction strands were changed toward uninterrupted and rounded meshwork loops. Functionally, the paracellular resistance of claudin-3-transfected monolayers was strongly elevated, causing an increase in transepithelial resistance compared to vector controls. Permeabilities for mono- and divalent cations and for anions were decreased. In the high-claudin-2 system, claudin-3 reduced claudin-2-induced cation selectivity, while in the low-claudin-2 system no charge preference was observed, the latter thus reflecting the "intrinsic" action of claudin-3. Furthermore, the passage of the paracellular tracers fluorescein (332Da) and FD-4 (4kDa) was decreased, whereas the permeability to water was not affected. We demonstrate that claudin-3 alters the tight junction meshwork and seals the paracellular pathway against the passage of small ions of either charge and uncharged solutes. Thus, in a kidney model epithelium, claudin-3 acts as a general barrier-forming protein.

  13. Altered distribution of tight junction proteins after intestinal ischaemia/reperfusion injury in rats

    PubMed Central

    Li, Qiurong; Zhang, Qiang; Wang, Chenyang; Liu, Xiaoxiang; Qu, Linlin; Gu, Lili; Li, Ning; Li, Jieshou

    2009-01-01

    Tight junction (TJ) disruptions have been demonstrated both in vitro and more recently in vivo in infection. However, the molecular basis for changes of TJ during ischaemia-reperfusion (I/R) injury is poorly understood. In the present study, intestinal damage was induced by I/R in an animal model. As assessed by TUNEL and propidium iodide uptake, we showed that I/R injury induced apoptosis as well as necrosis in rat colon, and the frequency of apoptotic and necrotic cells reached the maximum at 5 hrs of reperfusion. Immunofluorescence microscopy revealed that claudins 1, 3 and 5 are strongly expressed in the surface epithelial cells of the colon; however, labelling of all three proteins was present diffusely within cells and no longer focused at the lateral cell boundaries after I/R. Using Western blot analysis, we found that distribution of TJ proteins in membrane microdomains of TJ was markedly affected in I/R injury rats. Occludin, ZO-1, claudin-1 and claudin-3 were completely displaced from TX-100 insoluble fractions to TX-100 soluble fractions, and claudin-5 was partly displaced. The distribution of lipid raft marker protein caveolin-1 was also changed after I/R. I/R injury results in the disruption of TJs, which characterized by relocalization of the claudins 1, 3 and 5 and an increase in intestinal permeability using molecular tracer measurement. I/R injury altered distribution of TJ proteins in vivo that was associated with functional TJ deficiencies. PMID:19929946

  14. Reproductive age-related changes in the blood brain barrier: Expression of IgG and tight junction proteins

    PubMed Central

    Bake, Shameena; Friedman, Jonathan A; Sohrabji, Farida

    2009-01-01

    We previously demonstrated that there is significantly greater transfer of intravenously-injected Evan’s blue dye into the forebrain of acyclic (reproductive senescent) females compared to young adult females, indicating that blood brain barrier permeability is compromised in the reproductive senescent forebrain. The present study examined brain IgG expression and microvessel tight junction proteins to assess ovarian age-related changes in microvascular permeability, and further compared young and senescent females with age-matched males to distinguish changes attributable to age and reproductive senescence. Blood brain barrier breakdown are often associated with increased extravasation of plasma proteins and high levels of immunoglobulin G (IgG) in brain. In the present study, IgG expression was dramatically increased in the hippocampus and thalamus, but not the hypothalamus of reproductive senescent females compared to young adult females. In males, IgG expression was increased in all these regions in middle aged animals (aged-matched to senescent females) as compared to young males (age-matched to the young adult females). Furthermore, the proportion of hippocampal microvessels with perivascular IgG immunoreactivity was significantly greater in reproductive senescent females as compared to young adult females, while middle aged males and young adult males did not differ. The tight junctions between adjacent microvascular endothelial cells regulated by transmembrane proteins such as claudin-5 and occludin play a critical role in maintaining the blood brain barrier integrity. Increased hippocampal IgG expression in senescent females was paralleled by poor junctional localization of the tight junction protein claudin-5 in hippocampal microvessels. However, there was no difference in hippocampal claudin-5 localization between young adult and middle aged males, indicating that dysregulation of this junctional protein was associated with ovarian aging. Parallel

  15. Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

    SciTech Connect

    Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

    2008-11-01

    Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.

  16. Tight Junction Proteins Claudin-1 and Occludin Control Hepatitis C Virus Entry and Are Downregulated during Infection To Prevent Superinfection ▿ §

    PubMed Central

    Liu, Shufeng; Yang, Wei; Shen, Le; Turner, Jerrold R.; Coyne, Carolyn B.; Wang, Tianyi

    2009-01-01

    A tight junction (TJ) protein, claudin-1 (CLDN1), was identified recently as a key factor for hepatitis C virus (HCV) entry. Here, we show that another TJ protein, occludin, is also required for HCV entry. Mutational study of CLDN1 revealed that its tight junctional distribution plays an important role in mediating viral entry. Together, these data support the model in which HCV enters liver cells from the TJ. Interestingly, HCV infection of Huh-7 hepatoma cells downregulated the expression of CLDN1 and occludin, preventing superinfection. The altered TJ protein expression may contribute to the morphological and functional changes observed in HCV-infected hepatocytes. PMID:19052094

  17. Molecular and cellular mechanisms of tight junction dysfunction in the irritable bowel syndrome.

    PubMed

    Cheng, Peng; Yao, Jianning; Wang, Chunfeng; Zhang, Lianfeng; Kong, Wuming

    2015-09-01

    The pathophysiological mechanisms of the irritable bowel syndrome (IBS), one of the most prevalent gastrointestinal disorders, are complex and have not been fully elucidated. The present study aimed to investigate the molecular and cellular mechanisms of tight junction (TJ) dysfunction in IBS. Intestinal tissues of IBS and non‑IBS patients were examined to observe cellular changes by cell chemical tracer electron microscopy and transmission electron microscopy, and intestinal claudin‑1 protein was detected by immunohistochemistry, western blot analysis and fluorescence quantitative polymerase chain reaction. Compared with the control group, TJ broadening and the tracer extravasation phenomenon were observed in the diarrhea‑predominant IBS group, and a greater number of neuroendocrine cells and mast cells filled with high‑density particles in the endocrine package pulp as well as a certain extent of vacuolization were present. The expression of claudin‑1 in diarrhea‑predominant IBS patients was decreased, while it was increased in constipation‑predominant IBS patients. In conclusion, the results of the present study indicated that changes in cellular structure and claudin‑1 levels were associated with Tjs in IBS.

  18. Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium.

    PubMed

    El-Hashash, Ahmed H K; Turcatel, Gianluca; Varma, Saaket; Berika, Mohamed; Al Alam, Denise; Warburton, David

    2012-09-01

    Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKCζ-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.

  19. Alix-mediated assembly of the actomyosin–tight junction polarity complex preserves epithelial polarity and epithelial barrier

    PubMed Central

    Campos, Yvan; Qiu, Xiaohui; Gomero, Elida; Wakefield, Randall; Horner, Linda; Brutkowski, Wojciech; Han, Young-Goo; Solecki, David; Frase, Sharon; Bongiovanni, Antonella; d'Azzo, Alessandra

    2016-01-01

    Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin–tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical–basal polarity and in the maintenance of the epithelial barrier. PMID:27336173

  20. An induced junction photovoltaic cell

    NASA Technical Reports Server (NTRS)

    Call, R. L.

    1974-01-01

    Silicon solar cells operating with induced junctions rather than diffused junctions have been fabricated and tested. Induced junctions were created by forming an inversion layer near the surface of the silicon by supplying a sheet of positive charge above the surface. Measurements of the response of the inversion layer cell to light of different wavelengths indicated it to be more sensitive to the shorter wavelengths of the sun's spectrum than conventional cells. The greater sensitivity occurs because of the shallow junction and the strong electric field at the surface.

  1. Overexpression of caveolin-1 attenuates brain edema by inhibiting tight junction degradation

    PubMed Central

    Choi, Kang-Ho; Lee, Eun-Bin; Lee, Jung-Kil; Kim, Joon-Tae; Kim, Ja-Hae; Lee, Min-Cheol; Lee, Hong-Joon; Cho, Ki-Hyun

    2016-01-01

    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. PMID:27708218

  2. 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.

  3. Study on the pressure self-adaptive water-tight junction box in underwater vehicle

    NASA Astrophysics Data System (ADS)

    Huang, Haocai; Ye, Yanying; Leng, Jianxing; Yuan, Zhuoli; Chen, Ying

    2012-09-01

    Underwater vehicles play a very important role in underwater engineering. Water-tight junction box (WJB) is one of the key components in underwater vehicle. This paper puts forward a pressure self-adaptive water-tight junction box (PSAWJB) which improves the reliability of the WJB significantly by solving the sealing and pressure problems in conventional WJB design. By redundancy design method, the pressure self-adaptive equalizer (PSAE) is designed in such a way that it consists of a piston pressure-adaptive compensator (PPAC) and a titanium film pressureadaptive compensator (TFPAC). According to hydro-mechanical simulations, the operating volume of the PSAE is more than or equal to 11.6 % of the volume of WJB liquid system. Furthermore, the required operating volume of the PSAE also increases as the gas content of oil, hydrostatic pressure or temperature difference increases. The reliability of the PSAWJB is proved by hyperbaric chamber tests

  4. Clostridium difficile Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins

    PubMed Central

    Nusrat, A.; von Eichel-Streiber, C.; Turner, J. R.; Verkade, P.; Madara, J. L.; Parkos, C. A.

    2001-01-01

    The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021–16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting “raft-like” membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis. PMID:11179295

  5. The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

    PubMed Central

    Kwong, Raymond W. M.; Kumai, Yusuke; Perry, Steve F.

    2013-01-01

    Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (Ku) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H+-ATPase-rich cells or Na+/K+-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca2+-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased. PMID:23967101

  6. The role of aquaporin and tight junction proteins in the regulation of water movement in larval zebrafish (Danio rerio).

    PubMed

    Kwong, Raymond W M; Kumai, Yusuke; Perry, Steve F

    2013-01-01

    Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K(u)) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

  7. Escherichia coli STb enterotoxin dislodges claudin-1 from epithelial tight junctions.

    PubMed

    Nassour, Hassan; Dubreuil, J Daniel

    2014-01-01

    Enterotoxigenic Escherichia coli produce various heat-labile and heat-stable enterotoxins. STb is a low molecular weight heat-resistant toxin responsible for diarrhea in farm animals, mainly young pigs. A previous study demonstrated that cells having internalized STb toxin induce epithelial barrier dysfunction through changes in tight junction (TJ) proteins. These modifications contribute probably to the diarrhea observed. To gain insight into the mechanism of increased intestinal permeability following STb exposure we treated human colon cells (T84) with purified STb toxin after which cells were harvested and proteins extracted. Using a 1% Nonidet P-40-containing solution we investigated the distribution of claudin-1, a major structural and functional TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated.

  8. 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.

  9. Loss of tight junction proteins (Claudin 1, 4, and 7) correlates with aggressive behavior in colorectal carcinoma

    PubMed Central

    Süren, Dinç; Yıldırım, Mustafa; Kaya, Vildan; Alikanoğlu, Arsenal Sezgin; Bülbüller, Nurullah; Yıldız, Mustafa; Sezer, Cem

    2014-01-01

    Background Tight junction proteins in the cell organize paracellular permeability and they play a critical role in apical cell-to-cell adhesion and epithelial polarity. Claudins are major integral membrane proteins of tight junctions, especially Claudin 1, 4, and 7, which are known as the impermeability Claudins. In this study, we investigated the importance of loss of Claudin 1, 4, and 7 expression, and their relation to tumor progression in colorectal cancer patients. Material/Methods Loss of Claudin 1, 4, and 7 expression was examined by immunohistochemical method in 70 patients diagnosed with colorectal cancer. Cases with loss of Claudin expression in <1/3 of tumor cells were classified as mild loss, whereas cases with loss of Claudin expression ≥1/3 of tumor cells were classified as moderate-to-marked loss in order to evaluate the relation between loss of Claudin 1, 4, and 7 expression and clinicopathologic data. Results The severe suppression of Claudin 1, 4, and 7 expression was found to be significantly related to the depth of tumor invasion, positive regional lymph nodes, histological grade, lymphovascular invasion, perineural invasion, and lymphocytic response. Additionally, severity of loss in Claudin 4 expression was found to have a relation with distant metastasis. Conclusions Claudin 1, 4, and 7 are important building blocks of paracellular adhesion molecules. Their decreased expression in colorectal cancer seems to have critical effects on cell proliferation, motility, invasion, and immune response against the tumor. PMID:25038829

  10. Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glands

    PubMed Central

    Peppi, M; Ghabriel, M N

    2004-01-01

    Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague–Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels. PMID:15447685

  11. Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

    PubMed

    Rincon-Heredia, Ruth; Flores-Benitez, David; Flores-Maldonado, Catalina; Bonilla-Delgado, José; García-Hernández, Vicky; Verdejo-Torres, Odette; Castillo, Aida M; Larré, Isabel; Poot-Hernández, Augusto C; Franco, Martha; Gariglio, Patricio; Reyes, José L; Contreras, Rubén G

    2014-01-01

    In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

  12. Abnormal endothelial tight junctions in active lesions and normal-appearing white matter in multiple sclerosis.

    PubMed

    Plumb, Jonnie; McQuaid, Stephen; Mirakhur, Meenakshi; Kirk, John

    2002-04-01

    Blood-brain barrier (BBB) breakdown, demonstrable in vivo by enhanced MRI is characteristic of new and expanding inflammatory lesions in relapsing-remitting and chronic progressive multiple sclerosis (MS). Subtle leakage may also occur in primary progressive MS. However, the anatomical route(s) of BBB leakage have not been demonstrated. We investigated the possible involvement of interendothelial tight junctions (TJ) by examining the expression of TJ proteins (occludin and ZO-1 ) in blood vessels in active MS lesions from 8 cases of MS and in normal-appearing white (NAWM) matter from 6 cases. Blood vessels (10-50 per frozen section) were scanned using confocal laser scanning microscopy to acquire datasets for analysis. TJ abnormalities manifested as beading, interruption, absence or diffuse cytoplasmic localization of fluorescence, or separation of junctions (putative opening) were frequent (affecting 40% of vessels) in oil-red-O-positive active plaques but less frequent in NAWM (15%), and in normal (< 2%) and neurological controls (6%). Putatively "open" junctions were seen in vessels in active lesions and in microscopically inflamed vessels in NAWM. Dual fluorescence revealed abnormal TJs in vessels with pre-mortem serum protein leakage. Abnormal or open TJs, associated with inflammation may contribute to BBB leakage in enhancing MRI lesions and may also be involved in subtle leakage in non-enhancing focal and diffuse lesions in NAWM. BBB disruption due to tight junctional pathology should be regarded as a significant form of tissue injury in MS, alongside demyelination and axonopathy.

  13. The Role of Circulating Tight Junction Proteins in Evaluating Blood Brain Barrier Disruption following Intracranial Hemorrhage.

    PubMed

    Jiao, Xiaoyang; He, Ping; Li, Yazhen; Fan, Zhicheng; Si, Mengya; Xie, Qingdong; Chang, Xiaolan; Huang, Dongyang

    2015-01-01

    Brain injury after intracranial hemorrhage (ICH) results in significant morbidity and mortality. Blood brain barrier (BBB) disruption is a hallmark of ICH-induced brain injury; however, data mirroring BBB disruption in human ICH are scarce. The aim of this study was to assess the significance of circulating biomarkers in evaluating BBB disruption after ICH. Twenty-two patients with ICH were recruited in this study. Concentrations of the tight junction proteins (TJs) Claudin-5 (CLDN5), Occludin (OCLN), and zonula occludens 1 (ZO-1) and vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were measured by using enzyme-linked immunosorbent assay in serum and cerebrospinal fluid (CSF) samples obtained from patients with ICH. The white blood cell (WBC) count in blood and CSF, albumin (ALB) levels in the CSF (ALBCSF), and the BBB ratio were significantly higher in the ICH than in controls (p < 0.05). Significantly higher levels of CLDN5, OCLN, ZO-1, MMP-9, and VEGF in CSF were observed in the ICH group; these biomarkers were also positively associated with BBB ratio (p < 0.05). Our data revealed that circulating TJs could be considered the potential biomarkers reflecting the integrity of the BBB in ICH.

  14. The Role of Circulating Tight Junction Proteins in Evaluating Blood Brain Barrier Disruption following Intracranial Hemorrhage

    PubMed Central

    Jiao, Xiaoyang; He, Ping; Li, Yazhen; Fan, Zhicheng; Si, Mengya; Xie, Qingdong; Chang, Xiaolan; Huang, Dongyang

    2015-01-01

    Brain injury after intracranial hemorrhage (ICH) results in significant morbidity and mortality. Blood brain barrier (BBB) disruption is a hallmark of ICH-induced brain injury; however, data mirroring BBB disruption in human ICH are scarce. The aim of this study was to assess the significance of circulating biomarkers in evaluating BBB disruption after ICH. Twenty-two patients with ICH were recruited in this study. Concentrations of the tight junction proteins (TJs) Claudin-5 (CLDN5), Occludin (OCLN), and zonula occludens 1 (ZO-1) and vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were measured by using enzyme-linked immunosorbent assay in serum and cerebrospinal fluid (CSF) samples obtained from patients with ICH. The white blood cell (WBC) count in blood and CSF, albumin (ALB) levels in the CSF (ALBCSF), and the BBB ratio were significantly higher in the ICH than in controls (p < 0.05). Significantly higher levels of CLDN5, OCLN, ZO-1, MMP-9, and VEGF in CSF were observed in the ICH group; these biomarkers were also positively associated with BBB ratio (p < 0.05). Our data revealed that circulating TJs could be considered the potential biomarkers reflecting the integrity of the BBB in ICH. PMID:26586924

  15. Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex.

    PubMed

    Hou, Jianghui; Renigunta, Aparna; Konrad, Martin; Gomes, Antonio S; Schneeberger, Eveline E; Paul, David L; Waldegger, Siegfried; Goodenough, Daniel A

    2008-02-01

    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.

  16. PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function*

    PubMed Central

    Chen, Ying; Zhang, Hai-Sheng; Fong, Guo-Hua; Xi, Qiu-Lei; Wu, Guo-Hao; Bai, Chen-Guang; Ling, Zhi-Qiang; Fan, Li; Xu, Yi-Ming; Qin, Yan-Qing; Yuan, Tang-Long; Sun, Heng; Fang, Jing

    2015-01-01

    Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD. PMID:26124271

  17. Tight junction regulation by morphine and HIV-1 tat modulates blood-brain barrier permeability.

    PubMed

    Mahajan, Supriya D; Aalinkeel, Ravikumar; Sykes, Donald E; Reynolds, Jessica L; Bindukumar, B; Fernandez, Stanley F; Chawda, Ramnik; Shanahan, Thomas C; Schwartz, Stanley A

    2008-09-01

    Human immunodeficiency virus (HIV)-1 patients who abuse opiates are at a greater risk of developing neurological complications of AIDS. Alterations in blood-brain barrier (BBB) integrity are associated with cytoskeletal disorganization and disruption of tight junction (TJ) integrity. We hypothesize that opiates in combination with HIV-1 viral proteins can modulate TJ expression in primary brain microvascular endothelial cells (BMVEC), thereby compromising BBB integrity and exacerbating HIV-1 neuropathogenesis. We investigated the effect of morphine and/or tat on the expression of TJ proteins ZO-1, JAM-2, Occludin and P-glycoprotein and the functional effects of TJ modulation in BMVEC. Morphine and/or tat, via the activation of pro-inflammatory cytokines, intracellular Ca(2+) release, and activation of myosin light chain kinase, modulated TJ expression resulting in decreased transendothelial electric resistance and enhanced transendothelial migration across the BBB. These studies may lead to the development of novel anti-HIV-1 therapeutics that target specific TJ proteins, thus preventing TJ disruption in opiate using HIV-1 patients.

  18. The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis.

    PubMed

    Raschperger, Elisabeth; Thyberg, Johan; Pettersson, Sven; Philipson, Lennart; Fuxe, Jonas; Pettersson, Ralf F

    2006-05-15

    The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.

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

    SciTech Connect

    Imamura, Masafumi; Kojima, Takashi . E-mail: ktakashi@sapmed.ac.jp; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    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 in 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.

  20. AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

    PubMed Central

    Aznar, Nicolas; Patel, Arjun; Rohena, Cristina C; Dunkel, Ying; Joosen, Linda P; Taupin, Vanessa; Kufareva, Irina; Farquhar, Marilyn G; Ghosh, Pradipta

    2016-01-01

    Loss of epithelial polarity impacts organ development and function; it is also oncogenic. AMPK, a key sensor of metabolic stress stabilizes cell-cell junctions and maintains epithelial polarity; its activation by Metformin protects the epithelial barrier against stress and suppresses tumorigenesis. How AMPK protects the epithelium remains unknown. Here, we identify GIV/Girdin as a novel effector of AMPK, whose phosphorylation at a single site is both necessary and sufficient for strengthening mammalian epithelial tight junctions and preserving cell polarity and barrier function in the face of energetic stress. Expression of an oncogenic mutant of GIV (cataloged in TCGA) that cannot be phosphorylated by AMPK increased anchorage-independent growth of tumor cells and helped these cells to evade the tumor-suppressive action of Metformin. This work defines a fundamental homeostatic mechanism by which the AMPK-GIV axis reinforces cell junctions against stress-induced collapse and also provides mechanistic insight into the tumor-suppressive action of Metformin. DOI: http://dx.doi.org/10.7554/eLife.20795.001 PMID:27813479

  1. Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins.

    PubMed

    Chen, Xin; Oshima, Tadayuki; Tomita, Toshihiko; Fukui, Hirokazu; Watari, Jiro; Matsumoto, Takayuki; Miwa, Hiroto

    2011-08-01

    Experimental models for esophageal epithelium in vitro either suffer from poor differentiation or complicated culture systems. An air-liquid interface system with normal human bronchial epithelial cells can serve as a model of esophageal-like squamous epithelial cell layers. Here, we explore the influence of bile acids on barrier function and tight junction (TJ) proteins. The cells were treated with taurocholic acid (TCA), glycocholic acid (GCA), or deoxycholic acid (DCA) at different pH values, or with pepsin. Barrier function was measured by transepithelial electrical resistance (TEER) and the diffusion of paracellular tracers (permeability). The expression of TJ proteins, including claudin-1 and claudin-4, was examined by Western blotting of 1% Nonidet P-40-soluble and -insoluble fractions. TCA and GCA dose-dependently decreased TEER and increased paracellular permeability at pH 3 after 1 h. TCA (4 mM) or GCA (4 mM) did not change TEER and permeability at pH 7.4 or pH 4. The combination of TCA and GCA at pH 3 significantly decreased TEER and increased permeability at lower concentrations (2 mM). Pepsin (4 mg/ml, pH 3) did not have any effect on barrier function. DCA significantly decreased the TEER and increased permeability at pH 6, a weakly acidic condition. TCA (4 mM) and GCA (4 mM) significantly decreased the insoluble fractions of claudin-1 and claudin-4 at pH 3. In conclusion, acidic bile salts disrupted the squamous epithelial barrier function partly by modulating the amounts of claudin-1 and claudin-4. These results provide new insights for understanding the role of TJ proteins in esophagitis.

  2. Endothelial Tight Junctions Are Opened in Cholinergic-Evoked Salivation In Vivo.

    PubMed

    Cong, X; Zhang, Y; He, Q H; Wei, T; Zhang, X M; Zhang, J Z; Xiang, R L; Yu, G Y; Wu, L L

    2017-01-01

    Blood vessels provide the original supplies for the formation of primary saliva, which is regulated by the tight junctions (TJs) between endothelial cells. Previous studies have shown that blood flow increases with vasodilatation during cholinergic-evoked salivation. However, changes in vascular paracellular permeability and the role of endothelial TJs in salivation are unknown. Here, we established an in vivo paracellular permeability detection system and observed that the endothelial TJs were permeable to 4-kDa fluorescein isothiocyanate (FITC)-dextran while impermeable to 40- and 70-kDa FITC-dextran under an unstimulated condition in mouse submandibular glands (SMGs). Pilocarpine increased the flux of 4- and 40-kDa FITC-dextran out of blood vessels but did not affect 70-kDa FITC-dextran. Claudin 5, a TJ protein specifically localized in salivary endothelial cells, was redistributed from the apicolateral membranes to the lateral and basolateral membranes and cytoplasm in cholinergic-stimulated mouse SMGs and freshly cultured human SMG tissues. In the transplanted SMGs from epiphora patients, we found that claudin 5 was present in the basolateral membranes and cytoplasm, instead of the apical region in control SMGs. Moreover, the level of phospho-myosin light chain 2 increased within the blood vessels of the pilocarpine-stimulated mouse SMGs and transplanted human SMGs, while the downstream molecule F-actin was reorganized in the endothelial cells of the transplanted human SMGs. Taken together, our findings provide direct visual evidence that the opening of endothelial TJs and the redistribution of claudin 5 are essential events contributing to cholinergic-evoked salivation, thus enriching our understanding of the secretory mechanisms that link blood flow to primary saliva formation by regulating the endothelial paracellular permeability.

  3. β1-Na(+),K(+)-ATPase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury.

    PubMed

    Lin, X; Barravecchia, M; Kothari, P; Young, J L; Dean, D A

    2016-06-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with diverse disorders and characterized by disruption of the alveolar-capillary barrier, leakage of edema fluid into the lung, and substantial inflammation leading to acute respiratory failure. Gene therapy is a potentially powerful approach to treat ALI/ARDS through repair of alveolar epithelial function. Herein, we show that delivery of a plasmid expressing β1-subunit of the Na(+),K(+)-ATPase (β1-Na(+),K(+)-ATPase) alone or in combination with epithelial sodium channel (ENaC) α1-subunit using electroporation not only protected from subsequent lipopolysaccharide (LPS)-mediated lung injury, but also treated injured lungs. However, transfer of α1-subunit of ENaC (α1-ENaC) alone only provided protection benefit rather than treatment benefit although alveolar fluid clearance had been remarkably enhanced. Gene transfer of β1-Na(+),K(+)-ATPase, but not α1-ENaC, not only enhanced expression of tight junction protein zona occludins-1 (ZO-1) and occludin both in cultured cells and in mouse lungs, but also reduced pre-existing increase of lung permeability in vivo. These results demonstrate that gene transfer of β1-Na(+),K(+)-ATPase upregulates tight junction formation and therefore treats lungs with existing injury, whereas delivery of α1-ENaC only maintains pre-existing tight junction but not for generation. This indicates that the restoration of epithelial/endothelial barrier function may provide better treatment of ALI/ARDS.

  4. Tight Junction Proteins Claudin-3 and Claudin-4 Control Tumor Growth and Metastases12

    PubMed Central

    Shang, Xiying; Lin, Xinjian; Alvarez, Edwin; Manorek, Gerald; Howell, Stephen B

    2012-01-01

    The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling. PMID:23097631

  5. Probing the cis-arrangement of prototype tight junction proteins claudin-1 and claudin-3.

    PubMed

    Milatz, Susanne; Piontek, Jörg; Schulzke, Jörg-Dieter; Blasig, Ingolf E; Fromm, Michael; Günzel, Dorothee

    2015-06-15

    Claudins form a large family of TJ (tight junction) proteins featuring four transmembrane segments (TM1-TM4), two extracellular loops, one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the TJ-free cell line HEK (human embryonic kidney)-293. Using FRET analysis, the proximity of claudin N- and C-termini integrated in homopolymeric strands composed of claudin-3 or of heteropolymeric strands composed of claudin-1 and claudin-3 were determined. The main results are that (i) within homo- and heteropolymers, the average distance between the cytoplasmic ends of the TM1s of cis-interacting claudin molecules is shorter than the average distance between their TM4s, and (ii) TM1 segments of neighbouring claudins are oriented towards each other as the cytoplasmic end of TM1 is in close proximity to more other TM1 segments than TM4 is to other TM4 segments. The results indicate at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers. The data provide novel insight into the molecular TJ architecture consistent with a model with an antiparallel double-row cis-arrangement of classic claudin protomers within strands.

  6. Tight Junction Proteins Claudin-2 and -12 Are Critical for Vitamin D-dependent Ca2+ Absorption between Enterocytes

    PubMed Central

    Fujita, Hiroki; Sugimoto, Kotaro; Inatomi, Shuichiro; Maeda, Toshihiro; Osanai, Makoto; Uchiyama, Yasushi; Yamamoto, Yoko; Wada, Takuro; Kojima, Takashi; Yokozaki, Hiroshi; Yamashita, Toshihiko; Kato, Shigeaki; Sawada, Norimasa

    2008-01-01

    Ca2+ is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], is known to promote intestinal Ca2+ absorption. However, the molecules driving the paracellular Ca2+ absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca2+ transport and that expression of these claudins could be induced by 1α,25(OH)2D3. Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca2+ absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1α,25(OH)2D3 through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca2+ channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis. PMID:18287530

  7. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation.

    PubMed

    Tornavaca, Olga; Chia, Minghao; Dufton, Neil; Almagro, Lourdes Osuna; Conway, Daniel E; Randi, Anna M; Schwartz, Martin A; Matter, Karl; Balda, Maria S

    2015-03-16

    Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.

  8. The Interaction of JRAB/MICAL-L2 with Rab8 and Rab13 Coordinates the Assembly of Tight Junctions and Adherens Junctions

    PubMed Central

    Yamamura, Rie; Nishimura, Noriyuki; Nakatsuji, Hiroyoshi; Arase, Seiji

    2008-01-01

    The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the PM by using a Ca2+-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C) inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13 competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs. PMID:18094055

  9. Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli

    PubMed Central

    Ugalde-Silva, Paul; Gonzalez-Lugo, Octavio; Navarro-Garcia, Fernando

    2016-01-01

    The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption. PMID:27606286

  10. Dendrobium chrysotoxum Lindl. Alleviates Diabetic Retinopathy by Preventing Retinal Inflammation and Tight Junction Protein Decrease

    PubMed Central

    Yu, Zengyang; Gong, Chenyuan; Lu, Bin; Yang, Li; Sheng, Yuchen; Ji, Lili; Wang, Zhengtao

    2015-01-01

    Diabetic retinopathy (DR) is a serious complication of diabetes mellitus. This study aimed to observe the alleviation of the ethanol extract of Dendrobium chrysotoxum Lindl. (DC), a traditional Chinese herbal medicine, on DR and its engaged mechanism. After DC (30 or 300 mg/kg) was orally administrated, the breakdown of blood retinal barrier (BRB) in streptozotocin- (STZ-) induced diabetic rats was attenuated by DC. Decreased retinal mRNA expression of tight junction proteins (including occludin and claudin-1) in diabetic rats was also reversed by DC. Western blot analysis and retinal immunofluorescence staining results further confirmed that DC reversed the decreased expression of occludin and claudin-1 proteins in diabetic rats. DC reduced the increased retinal mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor α (TNFα), interleukin- (IL-) 6, and IL-1β in diabetic rats. In addition, DC alleviated the increased 1 and phosphorylated p65, IκB, and IκB kinase (IKK) in diabetic rats. DC also reduced the increased serum levels of TNFα, interferon-γ (IFN-γ), IL-6, IL-1β, IL-8, IL-12, IL-2, IL-3, and IL-10 in diabetic rats. Therefore, DC can alleviate DR by inhibiting retinal inflammation and preventing the decrease of tight junction proteins, such as occludin and claudin-1. PMID:25685822

  11. Electrophysiological characterization of tight junctional pathway of rabbit cornea treated with ophthalmic ingredients.

    PubMed

    Nakamura, Tadahiro; Yamada, Mikiko; Teshima, Mugen; Nakashima, Mikiro; To, Hideto; Ichikawa, Nobuhiro; Sasaki, Hitoshi

    2007-12-01

    The purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctional permeabilities of FITC-dextran 4,400 (FD-4) was also determined through the cornea in the presence of ophthalmic ingredients. Intact cornea showed approximately one k-ohmxcm(2) of transepithelial electrical resistance (TEER) and extremely low permeability of FD-4. The ophthalmic ingredients used in the present study were benzalkonium chloride (BK; 0.002%, 0.01%, 0.05%), ethylenediaminetetraacetic acid (EDTA; 0.5%), capric acid (C10; 0.25%), saponin (SP; 0.1%), taurocholic acid (TA; 1.0%) and sodium dodecyl sulfate (SDS; 0.01%). They were previously reported to be effective on corneal penetrations of various drugs at those concentrations without severe toxicity. These ingredients decreased TEER and increased corneal permeability of FD-4. BK reduced TEER in a concentration-dependent manner. There was a significant correlation (gamma=0.860) between the permeability coefficient (Papp) of FD-4 and conductance (Gm), which is the reciprocal value of TEER. It was also indicated that Papp and Gm have a relationship with the corneal cytotoxicity of the ingredients. In conclusion, an electrophysiological method using isolated cornea was very useful to determine the continuous and real-time influence of ophthalmic ingredients on the cornea. In this method, electrophysiological conductance must be able to predict corneal tight junction permeability and the corneal cytotoxicity of ingredients.

  12. Probiotics modify tight-junction proteins in an animal model of nonalcoholic fatty liver disease

    PubMed Central

    Briskey, David; Heritage, Mandy; Jaskowski, Lesley-Anne; Peake, Jonathan; Gobe, Glenda; Subramaniam, V. Nathan; Crawford, Darrell; Campbell, Catherine; Vitetta, Luis

    2016-01-01

    Background: We have investigated the effects of a multispecies probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high-fat diet or obesity-induced liver steatosis. Methods: Three groups of C57B1/6J mice were fed either a standard chow or a high-fat diet for 20 weeks, while a third group was fed a high-fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel samples were collected for analysis. Results: The expression of the tight-junction proteins ZO-1 and ZO-2 was reduced (p < 0.05) in high-fat diet-fed mice compared to chow-fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high-fat diet group (p < 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow-fed mice. Mice fed a high-fat diet ± probiotics had significant steatosis development compared with chow-fed mice (p < 0.05); steatosis was less severe in the probiotics group compared with the high-fat diet group. Hepatic triglyceride concentration was higher in mice fed a high-fat diet ± probiotics compared with the chow group (p < 0.05), and was lower in the probiotics group compared with the high-fat diet group (p < 0.05). Compared with chow-fed mice, serum glucose, cholesterol concentration and the activity of alanine transaminase were higher (p < 0.05), whereas serum triglyceride concentration was lower (p < 0.05) in mice fed a high-fat diet ± probiotics. Conclusions: Supplementation with a multispecies probiotic formulation helped to maintain tight-junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentration compared with a high-fat diet alone. PMID:27366215

  13. Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium.

    PubMed

    Hou, Jianghui; Renigunta, Aparna; Gomes, Antonio S; Hou, Mingli; Paul, David L; Waldegger, Siegfried; Goodenough, Daniel A

    2009-09-08

    Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16- and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.

  14. Altered expression of tight junction proteins and matrix metalloproteinases in thiamine-deficient mouse brain.

    PubMed

    Beauchesne, Elizabeth; Desjardins, Paul; Hazell, Alan S; Butterworth, Roger F

    2009-09-01

    Wernicke's encephalopathy (WE) in humans is a metabolic disorder caused by thiamine deficiency (TD). In both humans and experimental animals, TD leads to selective neuronal cell death in diencephalic and brainstem structures. Neuropathologic features of WE include petechial hemorrhagic lesions, and blood-brain barrier (BBB) breakdown has been suggested to play an important role in the pathogenesis of TD. The goal of the present study was to examine expression of the tight junction (TJ) protein occludin, its associated scaffolding proteins zona occludens (ZO-1 and ZO-2), and to measure matrix metalloproteinase (MMP) levels as a function of regional BBB permeability changes in thiamine-deficient mice. TD was induced in 12-week-old male C57Bl/6 mice by feeding a thiamine-deficient diet and administration of the central thiamine antagonist pyrithiamine. BBB permeability was measured by IgG extravasation; expression of occludin, ZO-1 and ZO-2 was measured by Western blot analysis and RT-PCR, structural integrity of the BBB was assessed using occludin and ZO-1 immunostaining, and MMPs levels were measured by gelatin zymography and immunohistochemistry. Studies were performed in vulnerable (medial thalamus) versus spared (frontal cortex) regions of the brain. Hemorrhagic lesions, selective increases in brain IgG extravasation, a concomitant loss in protein expression of occludin, ZO-1 and ZO-2, as well as decreased and disrupted patterns of occludin and ZO-1 immunostaining were observed in the medial thalamus of thiamine-deficient mice. MMP-9 levels were also selectively increased in the medial thalamus of these animals, and were found to be localized in the vascular endothelium, as well as in cells with an apparent polymorphonuclear morphology. No changes of TJ gene expression were observed. These results indicate that alterations in TJ proteins occur in TD, and offer a plausible explanation for the selective increase in BBB permeability in thiamine-deficient animals

  15. Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo

    PubMed Central

    Tietgens, Amber J.; Van Itallie, Christina M.; Vitale‐Cross, Lynn; Jarnik, Michal; Harding, Olivia J.; Anderson, James M.; Gutkind, J. Silvio; Weigert, Roberto; Arias, Irwin M.

    2016-01-01

    Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329) PMID:27396550

  16. Synergistic regulation of endothelial tight junctions by antioxidant (Se) and polyunsaturated lipid (GLA) via Claudin-5 modulation.

    PubMed

    Martin, Tracey A; Das, Tapas; Mansel, Robert E; Jiang, Wen G

    2006-08-01

    Tight junctions (TJs) in endothelial cells act as cell-cell adhesion structures, governing paracellular permeability (PCP). Disruption can lead to leaky vascular bed and potentially to oedema and swelling of tissues, the aetiology of mastalgia. These changes may also cause vascular spread of cancer cells. This study aimed to determine whether the function of TJs in endothelial cells can be strengthened by gamma linolenic acid (GLA), selenium (Se) and iodine (I) in the presence of 17beta-estradiol (17beta-estradiol), which causes leakage of endothelial cells by disruption of TJs in endothelium. GLA, I, and Se individually increased transendothelial resistance. The combination of all three agents also had a significant effect on TER. Addition of GLA/Se/I reduced PCP of the endothelial cells. Treatment with GLA/Se/I reversed the effect of 17beta-estradiol in reducing TER and increasing PCP. Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h there was increasing relocation to endothelial cell-cell junctions of the TJ proteins Claudin-5, Occludin, and ZO-1. Interestingly, this relocation was particularly evident with treatments containing I when probing with Claudin-5 and those containing Se for Occludin. There was a small increase in overall protein levels when examined by Western blotting after treatment with GLA/Se/I when probed with Claudin-5 and Occludin. We report that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells, by way of regulating the distribution of Claudin-5, Occludin, and ZO-1. Interestingly, this combination was also able to completely reverse the effect of 17beta-estradiol in these cells.

  17. 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.

  18. CD24 regulated gene expression and distribution of tight junction proteins is associated with altered barrier function in oral epithelial monolayers

    PubMed Central

    Ye, Ping; Nadkarni, Mangala A; Simonian, Mary; Hunter, Neil

    2009-01-01

    Background Control of intercellular penetration of microbial products is critical for the barrier function of oral epithelia. We demonstrated that CD24 is selectively and strongly expressed in the cells of the epithelial attachment to the tooth and the epithelial lining of the diseased periodontal pocket and studies in vitro showed that CD24 regulated expression of the epithelial intercellular adhesion protein E-cadherin. Results In the present study, the barrier function of oral epithelial cell monolayers to low molecular weight dextran was assayed as a model for the normal physiological function of the epithelial attachment to limit ingress of microbial products from oral microbial biofilms. Paracellular transfer of low molecular weight dextran across monolayers of oral epithelial cells was specifically decreased following incubation with anti-CD24 peptide antibody whereas passage of dextran across the monolayer was increased following silencing of mRNA for CD24. Changes in barrier function were related to the selective regulation of the genes encoding zonula occludens-1, zonula occludens-2 and occludin, proteins implicated in tight junctions. More particularly, enhanced barrier function was related to relocation of these proteins to the cell periphery, compatible with tight junctions. Conclusion CD24 has the constitutive function of maintaining expression of selected genes encoding tight junction components associated with a marginal barrier function of epithelial monolayers. Activation by binding of an external ligand to CD24 enhances this expression but is also effective in re-deployment of tight junction proteins that is aligned with enhanced intercellular barrier function. These results establish the potential of CD24 to act as a potent regulator of the intercellular barrier function of epithelia in response to local microbial ecology. PMID:19138432

  19. Tight junctions and paracellular fluid and ion transport in salivary glands.

    PubMed

    Zhang, Guo H; Wu, Li Ling; Yu, Guang Yan

    2013-01-01

    Primary saliva is formed by salivary epithelial endpieces through two pathways, the transcellular and the paracellular pathways. While the mechanisms of ion transport through the transcellular pathway have been well studied, our understanding of fluid and electrolyte transport through the paracellular pathway remains rudimentary. Increasing evidence indicates that the tight junction (TJ) proteins form and regulate the paracellular pathway, although other intercellular junctions are probably involved. The structure of the TJ is complex and has not been well characterised. A functioning TJ is formed by multiple proteins, including membrane, cytoplasmic scaffolding, and signalling proteins. Paracellular fluid and electrolyte flow is mediated by high-capacity, charge- and size-restrictive small pores with a radius of 4 to 6 Å, whereas macromolecules pass through low-capacity, nonrestrictive large pores. Although the characteristics of these pores need to be further delineated, it is clear that they are under the regulation of the autonomic nervous system, endocrine, paracrine and autocrine systems, and various pathological factors. To date, the majority of the evidence for paracellular fluid and ion transport is accumulated from the studies using various epithelia other than salivary glands. Further investigations to explore the structure, function, and regulation of the paracellular pathway in salivary epithelia are needed to better understand the mechanism of saliva secretion.

  20. ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum

    SciTech Connect

    Nighot, Prashant K.; Moeser, Adam J.; Ryan, Kathleen A.; Ghashghaei, Troy; Blikslager, Anthony T.

    2009-01-01

    Background and aims: Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2{sup -/-} mice. Methods: Wild type, ClC-2 heterozygous and ClC-2{sup -/-} murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of {sup 3}H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation. Results: Following ischemic injury, ClC-2{sup -/-} mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2{sup -/-} mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2{sup -/-} mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2{sup -/-} mice. Conclusions: These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.

  1. ZEB2-transgene expression in the epidermis compromises the integrity of the epidermal barrier through the repression of different tight junction proteins.

    PubMed

    Tatari, Marianthi N; De Craene, Bram; Soen, Bieke; Taminau, Joachim; Vermassen, Petra; Goossens, Steven; Haigh, Katharina; Cazzola, Silvia; Lambert, Jo; Huylebroeck, Danny; Haigh, Jody J; Berx, Geert

    2014-09-01

    Epithelial homeostasis within the epidermis is maintained by means of multiple cell-cell adhesion complexes such as adherens junctions, tight junctions, gap junctions, and desmosomes. These complexes co-operate in the formation and the regulation of the epidermal barrier. Disruption of the epidermal barrier through the deregulation of the above complexes is the cause behind a number of skin disorders such as psoriasis, dermatitis, keratosis, and others. During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their adhesive capacities and gain mesenchymal properties. ZEB transcription factors are key inducers of EMT. In order to gain a better understanding of the functional role of ZEB2 in epidermal homeostasis, we generated a mouse model with conditional overexpression of Zeb2 in the epidermis. Our analysis revealed that Zeb2 expression in the epidermis leads to hyperproliferation due to the combined downregulation of different tight junction proteins compromising the epidermal barrier. Using two epidermis-specific in vivo models and in vitro promoter assays, we identified occludin as a new Zeb2 target gene. Immunohistological analysis performed on human skin biopsies covering various pathogeneses revealed ZEB2 expression in the epidermis of pemphigus vulgaris. Collectively, our data support the notion for a potential role of ZEB2 in intracellular signaling of this disease.

  2. Effects of Interleukin-6 on the Expression of Tight Junction Proteins in Isolated Cerebral Microvessels from Yearling and Adult Sheep

    PubMed Central

    Cohen, Susan S.; Min, May; Cummings, Erin E.; Chen, Xiaodi; Sadowska, Grazyna B.; Sharma, Surendra; Stonestreet, Barbara S.

    2013-01-01

    Objectives The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions (TJs) are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of TJs. We hypothesized that IL-6 down-regulates key protein constituents of endothelial TJs (e.g., occludin and claudin-5). Methods We examined the effects of IL-6 on TJ protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate buffered saline), 1, 10, and 100 ng/mL for 24 hours. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. Results Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (P<0.05) occludin expression in microvessels from yearling and adult sheep, and claudin-5 in microvessels from adult sheep However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in TJ proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 pre-incubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. Conclusion We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep. PMID:23867217

  3. Mutations in the Tight-Junction Gene Claudin 19 (CLDN19) Are Associated with Renal Magnesium Wasting, Renal Failure, and Severe Ocular Involvement

    PubMed Central

    Konrad, Martin; Schaller, André; Seelow, Dominik; Pandey, Amit V.; Waldegger, Siegfried; Lesslauer, Annegret; Vitzthum, Helga; Suzuki, Yoshiro; Luk, John M.; Becker, Christian; Schlingmann, Karl P.; Schmid, Marcel; Rodriguez-Soriano, Juan; Ariceta, Gema; Cano, Francisco; Enriquez, Ricardo; Jüppner, Harald; Bakkaloglu, Sevcan A.; Hediger, Matthias A.; Gallati, Sabina; Neuhauss, Stephan C. F.; Nürnberg, Peter; Weber, Stefanie

    2006-01-01

    Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina. PMID:17033971

  4. The inhibition of COPII trafficking is important for intestinal epithelial tight junction disruption during enteropathogenic Escherichia coli and Citrobacter rodentium infection.

    PubMed

    Thanabalasuriar, Ajitha; Kim, Jinoh; Gruenheid, Samantha

    2013-01-01

    Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are bacterial pathogens that cause severe illnesses in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host intestinal cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium. NleA interacts with and inhibits the mammalian COPII complex, impairing cellular secretion; this interaction is required for bacterial virulence. Although diarrhea is a hallmark of EPEC, EHEC and C. rodentium infections, the underlying mechanisms are not well characterized. One of the essential functions of the intestine is to maintain a barrier between the lumen and submucosa. Tight junctions seal the space between adjacent epithelial cells creating this barrier. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by EPEC, EHEC, and C. rodentium could result in a loss of barrier function. In this study, we demonstrate that NleA mediated COPII inhibition is required for EPEC- and C. rodentium-mediated disruption of tight junction proteins and increases in fecal water content.

  5. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

    PubMed

    Markov, Alexander G; Falchuk, Evgeny L; Kruglova, Natalia M; Rybalchenko, Oksana V; Fromm, Michael; Amasheh, Salah

    2014-11-01

    Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Molecular Architecture of the Blood Brain Barrier Tight Junction Proteins--A Synergistic Computational and In Vitro Approach.

    PubMed

    Irudayanathan, Flaviyan Jerome; Trasatti, John P; Karande, Pankaj; Nangia, Shikha

    2016-01-14

    The blood-brain barrier (BBB) constituted by claudin-5 tight junctions is critical in maintaining the homeostasis of the central nervous system, but this highly selective molecular interface is an impediment for therapeutic interventions in neurodegenerative and neurological diseases. Therapeutic strategies that can exploit the paracellular transport remain elusive due to lack of molecular insights of the tight junction assembly. This study focuses on analyzing the membrane driven cis interactions of claudin-5 proteins in the formation of the BBB tight junctions. We have adopted a synergistic approach employing in silico multiscale dynamics and in vitro cross-linking experiments to study the claudin-5 interactions. Long time scale simulations of claudin-5 monomers, in seven different lipid compositions, show formation of cis dimers that subsequently aggregate into strands. In vitro formaldehyde cross-linking studies also conclusively show that cis-interacting claudin-5 dimers cross-link with short methylene spacers. Using this synergistic approach, we have identified five unique dimer interfaces in our simulations that correlate with the cross-linking experiments, four of which are mediated by transmembrane (TM) helices and the other mediated by extracellular loops (ECL). Potential of mean force calculations of these five dimers revealed that the TM mediated interfaces, which can have distinctive leucine zipper interactions in some cases, are more stable than the ECL mediated interface. Additionally, simulations show that claudin-5 dimerization is significantly influenced by the lipid microenvironment. This study captures the fundamental interactions responsible for the BBB tight junction assembly and offers a framework for extending this work to other tight junctions found in the body.

  8. Effects of di(2-ethylhexyl) phthalate on gap and tight junction protein expression in the testis of prepubertal rats.

    PubMed

    Sobarzo, Cristian M; Lustig, Livia; Ponzio, Roberto; Suescun, María Olga; Denduchis, Berta

    2009-11-01

    The aim of this study was to analyze whether di(2-ethylhexyl) phthalate (DEHP), a Sertoli and Leydig cell toxicant, is able to induce alterations in the expression of testicular gap and tight junction proteins. DEHP was administered by gavage (1 g/5 mL corn oil/kg body weight/day) to 25-day-old male Sprague-Dawley rats for 2 days (DEHP-27d) and control rats were treated with corn-oil vehicle for 2 days (C-27d); animals were killed 24 h after the last treatment. Testes of DEHP-27d rats showed different degrees of germ cell sloughing of seminiferous tubules (ST). No alterations of the blood testis barrier (BTB) by lanthanum tracer study were observed. ST of DEHP-27d rats showed a milder immunofluorescence and more restricted expression of connexin-43 (Cx43) in the adluminal and basal compartment compared to C-27d. In DEHP-27d rats, we found a discontinuous immunofluorescent (IF) pattern for zonula occludens (ZO-1), contrasting with the continuous IF profile observed in C-27d, and a delocalization of claudin-11. A decrease in Cx43 and ZO-1 and no changes in occludin expression were detected by Western blot in the testes of DEHP-27d rats. Results from 57-day-old rats treated with DEHP for 2 days and held for 30 days without treatment showed that the alterations in protein expression induced by DEHP are reversible. However, a delay of spermatogenesis compared to C-57d rats, occurred. Data demonstrated that DEHP does not impair BTB permeability but induces germ cell sloughing that might respond to a down regulation of Cx43 and ZO-1 that alters cell junction proteins.

  9. Curcumin prevents cisplatin-induced decrease in the tight and adherens junctions: relation to oxidative stress.

    PubMed

    Trujillo, Joyce; Molina-Jijón, Eduardo; Medina-Campos, Omar Noel; Rodríguez-Muñoz, Rafael; Reyes, José Luis; Loredo, María L; Barrera-Oviedo, Diana; Pinzón, Enrique; Rodríguez-Rangel, Daniela Saraí; Pedraza-Chaverri, José

    2016-01-01

    Curcumin is a polyphenol and cisplatin is an antineoplastic agent that induces nephrotoxicity associated with oxidative stress, apoptosis, fibrosis and decrease in renal tight junction (TJ) proteins. The potential effect of curcumin against alterations in TJ structure and function has not been evaluated in cisplatin-induced nephrotoxicity. The present study explored whether curcumin is able to prevent the cisplatin-induced fibrosis and decreased expression of the TJ and adherens junction (AJ) proteins occludin, claudin-2 and E-cadherin in cisplatin-induced nephrotoxicity. Curcumin (200 mg kg(-1)) was administered in three doses, and rats were sacrificed 72 h after cisplatin administration. Curcumin was able to scavenge, in a concentration-dependent way, superoxide anion, hydroxyl radical, peroxyl radical, singlet oxygen, peroxynitrite anion, hypochlorous acid and hydrogen peroxide. Cisplatin-induced renal damage was associated with alterations in plasma creatinine, expression of neutrophil gelatinase-associated lipocalin and of kidney injury molecule-1, histological damage, increase in apoptosis, fibrosis (evaluated by transforming growth factor β1, collagen I and IV and α-smooth muscle actin expressions), increase in oxidative/nitrosative stress (evaluated by Hsp70/72 expression, protein tyrosine nitration, superoxide anion production in isolated glomeruli and proximal tubules, and protein levels of NADPH oxidase subunits p47(phox) and gp91(phox), protein kinase C β2, and Nrf2) as well as by decreased expression of occludin, claudin-2, β-catenin and E-cadherin. Curcumin treatment prevented all the above-described alterations. The protective effect of curcumin against cisplatin-induced fibrosis and decreased proteins of the TJ and AJ was associated with the prevention of glomerular and proximal tubular superoxide anion production induced by NADPH oxidase activity.

  10. 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.

  11. Heat shock treatment protects osmotic stress–induced dysfunction of the blood-brain barrier through preservation of tight junction proteins

    PubMed Central

    Lu, Tzong-Shi; Chen, Hsiang-Wen; Huang, Maw-Hsiung; Wang, Shu-Jung; Yang, Rei-Cheng

    2004-01-01

    The blood-brain barrier (BBB) is a specialized structure in the central nervous system (CNS), which participates in maintenance of a state of cerebrospinal fluid homeostasis. The endothelial cells of the cerebral capillaries and the tight junctions between them form the basis of the BBB. Research has shown that destruction of the BBB is associated with diseases of the CNS. However, there is little research on how the BBB might be protected. In this study, we used a high osmotic solution (1.6 M d-mannitol) to open the BBB of rats and Evans blue dye as a macromolecular marker. The effect of heat shock treatment was evaluated. The results show that increased synthesis of heat shock protein 72 (Hsp72) was induced in the heated group only. BBB permeability was significantly less in the heat shock–treated group after hyperosmotic shock. The major tight junction proteins, occludin and zonula occludens (ZO)-1, were significantly decreased after d-mannitol treatment in the nonheated group, whereas they were preserved in the heated group. The coimmunoprecipitation studies demonstrated that Hsp72 could be detected in the precipitates of brain extract interacting with anti–ZO-1 antibodies as well as those interacting with anti–occludin antibodies in the heated group. We conclude that the integrity of tight junctions could be maintained by previous heat shock treatment, which might be associated with the increased production of Hsp72. PMID:15633295

  12. Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model

    PubMed Central

    2011-01-01

    Background Hematopoietic stem cell transplantation is increasingly performed for hematologic diseases. As a major side effect, acute graft versus host disease (GvHD) with serious gastrointestinal symptoms including diarrhea, gastrointestinal bleeding and high mortality can be observed. Because surveillance and biopsies of human gastrointestinal GvHD are difficult to perform, rare information of the alterations of the gastrointestinal barrier exists resulting in a need for systematic animal models. Methods To investigate the effects of GvHD on the intestinal barrier of the small intestine we utilized an established acute semi allogenic GvHD in C57BL/6 and B6D2F1 mice. Results By assessing the differential uptake of lactulose and mannitol in the jejunum, we observed an increased paracellular permeability as a likely mechanism for disturbed intestinal barrier function. Electron microscopy, immunohistochemistry and PCR analysis indicated profound changes of the tight-junction complex, characterized by downregulation of the tight junction protein occludin without any changes in ZO-1. Furthermore TNF-α expression was significantly upregulated. Conclusions This analysis in a murine model of GvHD of the small intestine demonstrates serious impairment of intestinal barrier function in the jejunum, with an increased permeability and morphological changes through downregulation and localization shift of the tight junction protein occludin. PMID:21977944

  13. Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4

    PubMed Central

    Jacob, Anne-Marie

    2012-01-01

    Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the “airway reopening” process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity. PMID:22898551

  14. Reversed cellular polarity in primary cutaneous mucinous carcinoma: A study on tight junction protein expression in sweat gland tumors.

    PubMed

    Nagasawa, Yusuke; Ishida-Yamamoto, Akemi

    2017-04-01

    Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO-1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot-like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.

  15. Hypoxia/Aglycemia-induced endothelial barrier dysfunction and tight junction protein downregulation can be ameliorated by citicoline.

    PubMed

    Ma, Xiaotang; Zhang, Huiting; 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.

  16. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

    PubMed

    Sen, Arnab; Sun, Rui; Krahn, Michael P

    2015-05-22

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

  17. Effects of flutamide in the rat testis on the expression of occludin, an integral member of the tight junctions.

    PubMed

    Gye, Myung Chan; Ohsako, Seiichiroh

    2003-07-20

    In an effort to uncover the gonadal impairment by the antiandrogen flutamide (FM) in males, the effect of subacute administration of FM on the expression of tight junction (TJ) genes that build the blood-testis barrier (BTB) was investigated in adult rat testis. At 13 weeks old of age male rats were given vehicle (corn oil) or FM (25 mg/kg per day, in corn oil) orally for 6 days. At 8 days (D8) after the first dose, testicular expression of the occludin, claudin-1, and -11 was analyzed by semiquantitative RT-PCR. The testicular weight of the FM-treated rats on D8 was a little but significantly higher than in the control group. On D8 the expression of occludin in the FM-treated animals was significantly decreased but claudin-1 and -11 were not altered significantly. Because FM administration inhibits germ cell differentiation, it is likely that the down-regulated occludin expression in FM-rat testes may be attributed to the alteration in the paracrine interaction between Sertoli cells and germ cells in testis. It also emphasized that FM might have differentially affected the transcription of TJ genes in Sertoli cells building the BTB. These findings provide a rationale for a number of observations on the gonadal impairment by FM in males and suggest that FM is potentially harmful to spermatogenesis by alteration of the BTB.

  18. Claudin-11 Tight Junctions in Myelin Are a Barrier to Diffusion and Lack Strong Adhesive Properties

    PubMed Central

    Denninger, Andrew R.; Breglio, Andrew; Maheras, Kathleen J.; LeDuc, Geraldine; Cristiglio, Viviana; Demé, Bruno; Gow, Alexander; Kirschner, Daniel A.

    2015-01-01

    The radial component is a network of interlamellar tight junctions (TJs) unique to central nervous system myelin. Ablation of claudin-11, a TJ protein, results in the absence of the radial component and compromises the passive electrical properties of myelin. Although TJs are known to regulate paracellular diffusion, this barrier function has not been directly demonstrated for the radial component, and some evidence suggests that the radial component may also mediate adhesion between myelin membranes. To investigate the physical properties of claudin-11 TJs, we compared fresh, unfixed Claudin 11-null and control nerves using x-ray and neutron diffraction. In Claudin 11-null tissue, we detected no changes in myelin structure, stability, or membrane interactions, which argues against the notion that myelin TJs exhibit significant adhesive properties. Moreover, our osmotic stressing and D2O-H2O exchange experiments demonstrate that myelin lacking claudin-11 is more permeable to water and small osmolytes. Thus, our data indicate that the radial component serves primarily as a diffusion barrier and elucidate the mechanism by which TJs govern myelin function. PMID:26445439

  19. Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

    PubMed

    Zhu, Jing-Cheng; Si, Meng-Ya; Li, Ya-Zhen; Chen, Huan-Zhu; Fan, Zhi-Cheng; Xie, Qing-Dong; Jiao, Xiao-Yang

    2016-03-21

    The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Enhanced nasal absorption of hydrophilic markers after dosing with AT1002, a tight junction modulator.

    PubMed

    Song, Keon-Hyoung; Fasano, Alessio; Eddington, Natalie D

    2008-05-01

    AT1002 is a six-mer synthetic peptide, H-FCIGRL-OH, that retains the delta G and Zot biological activity of reversibly opening tight junctions and increases the paracellular transport of drugs. The objective of this study was to evaluate the possible use of AT1002 in enhancing the nasal availability of macromolecules using large paracellular markers as model agents. Male Sprague-Dawley rats cannulated in the jugular vein were randomly assigned to receive radiolabelled paracellular markers, [14C]PEG4000 or [14C]inulin, with/without AT1002, for each intranasal study. The plasma concentration of PEG4000 with AT1002 (10mg/kg) was significantly higher than that from PEG4000 control over 360 min following intranasal administration. The AUC0-360 min and Cmax from the PEG4000/AT1002 (10mg/kg) treatment were statistically (p<0.05) increased to 235% and 357%, of control, respectively. When inulin was administered with AT1002 (10 mg/kg), the plasma concentration was significantly higher (p<0.05) than control over 360 min, and increases (p<0.05) of 292% and 315% for AUC0-360 min and Cmax over control were observed, respectively. AT1002 significantly increased the nasal absorption of molecular weight markers, PEG4000 and inulin. This study suggests that AT1002 may be used to enhance the systemic availability of macromolecules when administered concurrently.

  1. Adaptive evolution of tight junction protein claudin-14 in echolocating whales.

    PubMed

    Xu, Huihui; Liu, Yang; He, Guimei; Rossiter, Stephen J; Zhang, Shuyi

    2013-11-10

    Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats.

  2. Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm’s Canal Endothelia

    PubMed Central

    Tam, Lawrence C. S.; Reina-Torres, Ester; Sherwood, Joseph M.; Cassidy, Paul S.; Crosbie, Darragh E.; Lütjen-Drecoll, Elke; Flügel-Koch, Cassandra; Perkumas, Kristin; Humphries, Marian M.; Kiang, Anna-Sophia; O’Callaghan, Jeffrey; Callanan, John J.; Read, A. Thomas; Ethier, C. Ross; O’Brien, Colm; Lawrence, Matthew; Campbell, Matthew; Stamer, W. Daniel; Overby, Darryl R.; Humphries, Pete

    2017-01-01

    The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system. PMID:28091584

  3. CAR expression in human embryos and hESC illustrates its role in pluripotency and tight junctions.

    PubMed

    Krivega, M; Geens, M; Van de Velde, H

    2014-11-01

    Coxsackie virus and adenovirus receptor, CXADR (CAR), is present during embryogenesis and is involved in tissue regeneration, cancer and intercellular adhesion. We investigated the expression of CAR in human preimplantation embryos and embryonic stem cells (hESC) to identify its role in early embryogenesis and differentiation. CAR protein was ubiquitously present during preimplantation development. It was localised in the nucleus of uncommitted cells, from the cleavage stage up to the precursor epiblast, and corresponded with the presence of soluble CXADR3/7 splice variant. CAR was displayed on the membrane, involving in the formation of tight junction at compaction and blastocyst stages in both outer and inner cells, and CAR corresponded with the full-length CAR-containing transmembrane domain. In trophectodermal cells of hatched blastocysts, CAR was reduced in the membrane and concentrated in the nucleus, which correlated with the switch in RNA expression to the CXADR4/7 and CXADR2/7 splice variants. The cells in the outer layer of hESC colonies contained CAR on the membrane and all the cells of the colony had CAR in the nucleus, corresponding with the transmembrane CXADR and CXADR4/7. Upon differentiation of hESC into cells representing the three germ layers and trophoblast lineage, the expression of CXADR was downregulated. We concluded that CXADR is differentially expressed during human preimplantation development. We described various CAR expressions: i) soluble CXADR marking undifferentiated blastomeres; ii) transmembrane CAR related with epithelial-like cell types, such as the trophectoderm (TE) and the outer layer of hESC colonies; and iii) soluble CAR present in TE nuclei after hatching. The functions of these distinct forms remain to be elucidated.

  4. A Novel Perspective and Approach to Intestinal Octreotide Absorption: Sinomenine-Mediated Reversible Tight Junction Opening and Its Molecular Mechanism

    PubMed Central

    Li, Yuling; Duan, Zhijun; Tian, Yan; Liu, Zhen; Wang, Qiuming

    2013-01-01

    In this work, we assessed the effects of sinomenine (SN) on intestinal octreotide (OCT) absorption both in Caco-2 cell monolayers and in rats. We also investigated the molecular mechanisms of tight junction (TJ) disruption and recovery by SN-mediated changes in the claudin-1 and protein kinase C (PKC) signaling pathway. The data showed that exposure to SN resulted in a significant decrease in the expression of claudin-1, which represented TJ weakening and paracellular permeability enhancement. Then, the recovery of TJ after SN removal required an increase in claudin-1, which demonstrated the transient and reversible opening for TJ. Meanwhile, the SN-mediated translocation of PKC-α from the cytosol to the membrane was found to prove PKC activation. Finally, SN significantly improved the absolute OCT bioavailability in rats and the transport rate in Caco-2 cell monolayers. We conclude that SN has the ability to enhance intestinal OCT absorption and that these mechanisms are related at least in part to the important role of claudin-1 in SN-mediated, reversible TJ opening via PKC activation. PMID:23787475

  5. Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover.

    PubMed

    Gao, Fei; Artham, Sandeep; Sabbineni, Harika; Al-Azayzih, Ahmad; Peng, Xiao-Ding; Hay, Nissim; Adams, Ralf H; Byzova, Tatiana V; Somanath, Payaningal R

    2016-10-01

    Vascular permeability regulated by the vascular endothelial growth factor (VEGF) through endothelial-barrier junctions is essential for inflammation. Mechanisms regulating vascular permeability remain elusive. Although 'Akt' and 'Src' have been implicated in the endothelial-barrier regulation, it is puzzling how both agents that protect and disrupt the endothelial-barrier activate these kinases to reciprocally regulate vascular permeability. To delineate the role of Akt1 in endothelial-barrier regulation, we created endothelial-specific, tamoxifen-inducible Akt1 knockout mice and stable ShRNA-mediated Akt1 knockdown in human microvascular endothelial cells. Akt1 loss leads to decreased basal and angiopoietin1-induced endothelial-barrier resistance, and enhanced VEGF-induced endothelial-barrier breakdown. Endothelial Akt1 deficiency resulted in enhanced VEGF-induced vascular leakage in mice ears, which was rescued upon re-expression with Adeno-myrAkt1. Furthermore, co-treatment with angiopoietin1 reversed VEGF-induced vascular leakage in an Akt1-dependent manner. Mechanistically, our study revealed that while VEGF-induced short-term vascular permeability is independent of Akt1, its recovery is reliant on Akt1 and FoxO-mediated claudin expression. Pharmacological inhibition of FoxO transcription factors rescued the defective endothelial barrier due to Akt1 deficiency. Here we provide novel insights on the endothelial-barrier protective role of VEGF in the long term and the importance of Akt1-FoxO signaling on tight-junction stabilization and prevention of vascular leakage through claudin expression.

  6. Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain

    PubMed Central

    Mullier, Amandine; Bouret, Sébastien G.; Prevot, Vincent; Dehouck, Bénédicte

    2010-01-01

    The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we report on our use of immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1 and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. PMID:20127760

  7. Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain.

    PubMed

    Mullier, Amandine; Bouret, Sebastien G; Prevot, Vincent; Dehouck, Bénédicte

    2010-04-01

    The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties.

  8. Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia-induced bacterial growth.

    PubMed

    Patkee, Wishwanath R A; Carr, Georgina; Baker, Emma H; Baines, Deborah L; Garnett, James P

    2016-04-01

    Lung disease and elevation of blood glucose are associated with increased glucose concentration in the airway surface liquid (ASL). Raised ASL glucose is associated with increased susceptibility to infection by respiratory pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. We have previously shown that the anti-diabetes drug, metformin, reduces glucose-induced S. aureus growth across in vitro airway epithelial cultures. The aim of this study was to investigate whether metformin has the potential to reduce glucose-induced P. aeruginosa infections across airway epithelial (Calu-3) cultures by limiting glucose permeability. We also explored the effect of P. aeruginosa and metformin on airway epithelial barrier function by investigating changes in tight junction protein abundance. Apical P. aeruginosa growth increased with basolateral glucose concentration, reduced transepithelial electrical resistance (TEER) and increased paracellular glucose flux. Metformin pre-treatment of the epithelium inhibited the glucose-induced growth of P. aeruginosa, increased TEER and decreased glucose flux. Similar effects on bacterial growth and TEER were observed with the AMP activated protein kinase agonist, 5-aminoimidazole-4-carboxamide ribonucleotide. Interestingly, metformin was able to prevent the P. aeruginosa-induced reduction in the abundance of tight junction proteins, claudin-1 and occludin. Our study highlights the potential of metformin to reduce hyperglycaemia-induced P. aeruginosa growth through airway epithelial tight junction modulation, and that claudin-1 and occludin could be important targets to regulate glucose permeability across airway epithelia and supress bacterial growth. Further investigation into the mechanisms regulating metformin and P. aeruginosa action on airway epithelial tight junctions could yield new therapeutic targets to prevent/suppress hyperglycaemia-induced respiratory infections, avoiding the use of antibiotics.

  9. Epithelial fluid transport: protruding macromolecules and space charges can bring about electro-osmotic coupling at the tight junctions.

    PubMed

    Rubashkin, A; Iserovich, P; Hernández, J A; Fischbarg, J

    2005-12-01

    The purpose of the present work is to investigate whether the idea of epithelial fluid transport based on electro-osmotic coupling at the level of the leaky tight junction (TJ) can be further supported by a plausible theoretical model. We develop a model for fluid transport across epithelial layers based on electro-osmotic coupling at leaky tight junctions (TJ) possessing protruding macromolecules and fixed electrical charges. The model embodies systems of electro-hydrodynamic equations for the intercellular pathway, namely the Brinkman and the Poisson-Boltzmann differential equations applied to the TJ. We obtain analytical solutions for a system of these two equations, and are able to derive expressions for the fluid velocity profile and the electrostatic potential. We illustrate the model by employing geometrical parameters and experimental data from the corneal endothelium, for which we have previously reported evidence for a central role for electro-osmosis in translayer fluid transport. Our results suggest that electro-osmotic coupling at the TJ can account for fluid transport by the corneal endothelium. We conclude that electro-osmotic coupling at the tight junctions could represent one of the basic mechanisms driving fluid transport across some leaky epithelia, a process that remains unexplained.

  10. The MAPK/ERK-Signaling Pathway Regulates the Expression and Distribution of Tight Junction Proteins in the Mouse Proximal Epididymis.

    PubMed

    Kim, Bongki; Breton, Sylvie

    2016-01-01

    The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. The MAPK/ERK1/2 pathway is maintained active in the IS by testicular luminal factors and plays crucial roles in the maintenance and differentiation of the IS epithelium. Tight junctions (TJs) are constituents of the blood-epididymis barrier, which mediates the paracellular transport of ions, solutes, and water and controls epithelial cell differentiation, thereby contributing to the establishment of a unique luminal environment. We examine here the role of the MAPK/ERK1/2 pathway in the regulation of TJ proteins in the IS. Inhibition of mitogen activated protein kinase kinase (MAPKK or MEK1/2) with PD325901, followed by reduction of ERK1/2 phosphorylation (pERK), decreased zonula occludens (ZO)-2 expression and increased ZO-3 expression in TJs but had no effect on ZO-1 expression. In control mice, in addition to being located in TJs, claudin (Cldn)-1, Cldn-3, and Cldn-4 were detected in the basolateral membrane of epithelial cells, with enriched expression of Cldn-1 and Cldn-4 in basal cells. PD325901 reduced the expression of Cldn-1 and Cldn-4 at all locations without affecting Cldn-3. Occludin was undetectable in the IS of control mice, but PD325901 triggered its expression in TJs. No effect was observed for any of the proteins examined in the other epididymal regions. Our results indicate the participation of the MAPK/ERK1/2 pathway in the regulation of cell-cell events that control the formation and maintenance of the blood-epididymis barrier.

  11. 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.

  12. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery.

    PubMed

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-07-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

  13. Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery

    PubMed Central

    Yang, Yi; Thompson, Jeffrey F; Taheri, Saeid; Salayandia, Victor M; McAvoy, Thera A; Hill, Jeff W; Yang, Yirong; Estrada, Eduardo Y; Rosenberg, Gary A

    2013-01-01

    In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood–brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke. PMID:23571276

  14. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

    PubMed

    Furuse, M; Itoh, M; Hirase, T; Nagafuchi, A; Yonemura, S; Tsukita, S; Tsukita, S

    1994-12-01

    Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

  15. Escherichia coli heat-stable toxin b impairs intestinal epithelial barrier function by altering tight junction proteins.

    PubMed

    Ngendahayo Mukiza, Clément; Dubreuil, J Daniel

    2013-08-01

    Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.

  16. Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins

    PubMed Central

    Ngendahayo Mukiza, Clément

    2013-01-01

    Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea. PMID:23716609

  17. 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 CaV1.3 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.

  18. Utilizing Ultrasound to Transiently Increase Blood-Brain Barrier Permeability, Modulate of the Tight Junction Proteins, and Alter Cytoskeletal Structure.

    PubMed

    Bae, Mi Jung; Lee, Young Mi; Kim, Yeoun Hee; Han, Hyung Soo; Lee, Hak Jong

    2015-01-01

    The central nervous system is protected by the blood-brain barrier (BBB). The tight junction (TJ) proteins claudin-5 and zonula occludens-1 (ZO-1) as well as the cytoskeletal component F-actin play key roles in maintaining homeostasis of the BBB. Increases in BBB permeability may be beneficial for the delivery of pharmacological substances into the brain. Therefore, here, we assessed the use of ultrasound to induce transient enhancement of BBB permeability. We used fluorescein isothiocyanate (FITC)-dextran 40 to detect changes in the membrane permeability of bEnd.3 cells during ultrasound treatment. Ultrasound increased FITC-dextran 40 uptake into bEnd.3 cells for 2-6 h after treatment; however, normal levels returned after 24 h. An insignificant increase in lactate dehydrogenase (LDH) leakage also occurred 3 and 6 h after ultrasound treatment, whereas at 24 h, LDH leakage was indistinguishable between the control and treatment groups. Expression of claudin-5, ZO-1, and F-actin at the messenger RNA (mRNA) and protein levels was assessed with real-time polymerase chain reaction and western blotting. Ultrasound induced a transient decrease in claudin-5 mRNA and protein expression within 2 h of treatment; however, no significant changes in ZO-1 and F-actin expression were observed. Claudin-5, ZO-1, and F-actin immunofluorescence demonstrated that the cellular structures incorporating these proteins were transiently impaired by ultrasound. In conclusion, our ultrasound technique can temporarily increase BBB permeability without cytotoxicity to exposed cells, and the method can be exploited in the delivery of drugs to the brain with minimal damage.

  19. Different segments of the cerebral vasculature reveal specific endothelial specifications, while tight junction proteins appear equally distributed.

    PubMed

    Hanske, Sophie; Dyrna, Felix; Bechmann, Ingo; Krueger, Martin

    2017-04-01

    The identification of the "paucity of transportation vesicles" and "belt-like" tight junctions (TJs) of endothelial cells as the "morphological correlate of a blood-brain barrier" (BBB) by Reese and Karnovsky (J Cell Biol 34:207-217, 1967) has become textbook knowledge, and countless studies have helped to further define the elements, functions, and dynamics of the BBB. Most work, however, has focused on parenchymal capillaries or less clearly defined "microvessels", while a systematic study on similarities and differences between BBB architecture along the vascular tree within the brain and the meninges has been lacking. Since astrocytes induce endothelial cells to display BBB-typical characteristics by sonic hedgehog and Wnt/β-catenin signaling, we hypothesized that BBB-typical features should be most pronounced in parenchymal capillaries, where endothelium and astrocytes are separated by a basement membrane only. In contrast, this intimate contact is absent in leptomeningeal vessels, thereby potentially affecting BBB architecture. However, here, we show that claudin-3, claudin-5, zonula occludens-1, and occludin as typical constitutes of BBB TJs are comparably distributed in all segments of the parenchymal and the meningeal vascular tree of C57Bl6 mice. While electron microscopy revealed equally occluded interendothelial clefts, arterial vessels of the brain parenchyma but not within the meninges exhibited significantly longer TJ overlaps compared to capillaries. The highest density of endothelial vesicles was found in arterial vessels. Thus, endothelial expression of BBB-typical TJ proteins is not reflected by the distance to surrounding astrocytes, but electron microscopy reveals significant differences of endothelial specification along different segments of the CNS vasculature.

  20. Claudin 11 inter-sertoli tight junctions in the testis of the korean soft-shelled turtle (Pelodiscus maackii).

    PubMed

    Park, Chan Jin; Ha, Cheol Min; Lee, Jae Eun; Gye, Myung Chan

    2015-04-01

    Expression of claudin 11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testis. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the nonbreeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong, wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonula occludens 1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those during the nonbreeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the blood-testis barrier (BTB), where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJs expressing CLDN11 at the BTB in amniotes.

  1. Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium.

    PubMed

    Quesnell, Rebecca R; Erickson, Jamie; Schultz, Bruce D

    2007-01-01

    In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R(te)) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R(te) began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R(te). Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R(te), although total occludin was unchanged. Neither substitution of Na(+) with N-methyl-d-glucosamine (NMDG(+)) nor pharmacological inhibition of transcellular Na(+) transport pathways abrogated the effects of apical H-elec medium on R(te). Tumor necrosis factor alpha, but not interleukin-1beta nor interleukin-6, in the apical compartment caused a significant decrease in R(te) within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.

  2. Immunosuppressant MPA Modulates Tight Junction through Epigenetic Activation of MLCK/MLC-2 Pathway via p38MAPK

    PubMed Central

    Khan, Niamat; Pantakani, D. V. Krishna; Binder, Lutz; Qasim, Muhammad; Asif, Abdul R.

    2015-01-01

    Background: Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients, however, its use is also associated with adverse side effects like sporadic gastrointestinal (GI) disturbances. Recently, we reported the MPA induced tight junctions (TJs) deregulation which involves MLCK/MLC-2 pathway. Here, we investigated the global histone acetylation as well as gene-specific chromatin signature of several genes associated with TJs regulation in Caco-2 cells after MPA treatment. Results: The epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) at promoter regions of p38MAPK, ATF-2, MLCK, and MLC-2. In contrast, the promoter region of occludin was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin expression was negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be blocked by the inhibition of p38MAPK using a chemical inhibitor (SB203580). Conclusions: Collectively, our results highlight that MPA disrupts the structure of TJs via p38MAPK-dependent activation of MLCK/MLC-2 pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that p38MAPK-mediated lose integrity of epithelial monolayer could be the possible cause of GI disturbance (barrier dysfunction) in the intestine, leading to leaky style diarrhea observed in the organ-transplanted patients treated with MPA. PMID:26733876

  3. Perinatal administration of bisphenol A alters the expression of tight junction proteins in the uterus and reduces the implantation rate.

    PubMed

    Martínez-Peña, Annia A; Rivera-Baños, Jorge; Méndez-Carrillo, Laura L; Ramírez-Solano, Marcos I; Galindo-Bustamante, Aarón; Páez-Franco, J Carlos; Morimoto, Sumiko; González-Mariscal, Lorenza; Cruz, M Esther; Mendoza-Rodríguez, C Adriana

    2017-02-17

    We studied the effect of bisphenol-A (BPA) administration to rats, during the perinatal period, on the fertility of F1 generation and on the expression of tight junction (TJ) proteins in the uterus during early pregnancy. Pregnant Wistar dams (F0) received: BPA-L (0.05mg/kg/day), BPA-H (20mg/kg/day) or vehicle, from gestational day (GD) 6 to lactation day 21. F1 female pups were mated at 3 months of age and sacrificed at GD 1, 3, 6, and 7. Serum hormonal levels, ovulation rate, number of implantation sites and expression of TJ proteins in the uterus of F1 females were evaluated. BPA treatment induced no change in ovulation rate, but induced alterations in progesterone (P4) and estradiol (E2) serum levels, and in implantation rate. With regards to TJ proteins, BPA-H increased claudin-1 during all GDs; eliminated the peaks of claudins -3 and -4 at GD 3 and 6, respectively; and decreased claudin-7 at GD 6, ZO-1 from GD 1-6, and claudin-3 at GD 7 in stromal cells. BPA-L instead, eliminated claudin-3 peak at GD 3, increased claudin-4 and decreased claudin-7 from GD 1-6, decreased claudin-1 at GD 3 and 7 and claudin-4 at GD 7 in stromal cells. BPA-L also decreased ZO-1 at GDs 1 and 3 and increased ZO-1 at GD 6. Thus, BPA treatment during perinatal period perturbed, when the animals reached adulthood and became pregnant, the particular expression of TJ proteins in the uterine epithelium and reduced in consequence the number of implantation sites.

  4. Changes in the distribution pattern of Claudin tight junction proteins during the progression of mouse skin tumorigenesis

    PubMed Central

    Arabzadeh, Azadeh; Troy, Tammy-Claire; Turksen, Kursad

    2007-01-01

    Background Despite the fact that morphological and physiological observations suggest that the tight junction (TJ)-based permeability barrier is modified/disrupted in tumorigenesis, the role of members of the Claudin (Cldn) family of TJ proteins is not well-understood. Using a well-established two-stage chemical carcinogenesis model, we investigated the temporal and spatial changes in expression of those Cldns that we have previously demonstrated to be important in epidermal differentiation and the formation of the epidermal permeability barrier, i.e., Cldn1, Cldn6, Cldn11, Cldn12 and Cldn18. Methods The lower dorsal backskin of mice was treated topically with 7,12-dimethylbenz(a)anthracene (DMBA; 0.25 mg/ml in acetone) and following a 10-day incubation period, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 μg/ml in acetone) was applied three times a week to the same area. Backskin samples were dissected 2, 4, 6, 8 and 12 weeks after the initiation of the experimental protocol and immunohistochemistry was performed on sections using antibodies against the following: Cldn1, Cldn6, Cldn11, Cldn12, Cldn18, Ki67 and CD3. Results Our data indicate that along with the changes in epidermal cell morphology and differentiation that occur during tumor formation, there is a dramatic change in Cldn distribution consistent with cell polarity and barrier selectivity changes. Specifically, in the early stages of DMBA/TPA treatment, the suprabasal-specific Cldns occupy an expanded zone of expression corresponding to an increased number of suprabasal epidermal cell layers. As tumorigenesis progressed, the number of suprabasal epidermal layers positive for Cldn6, Cldn11, Cldn12 and Cldn18 was reduced, especially in the lower strata of the expanded suprabasal zone. In addition, a variably reduced cell membrane association of those differentiation-specific Cldns was observed, especially within the infiltrating epidermal structures. In contrast, Cldn1 (which is normally expressed in

  5. Carbon Monoxide-Releasing Molecule-2 Reduces Intestinal Epithelial Tight-Junction Damage and Mortality in Septic Rats

    PubMed Central

    Wang, Xin; Shi, Qiankun; Wang, Xiang; Yuan, Shoutao; Wang, Guozheng; Ji, Zhenling

    2015-01-01

    Objective Damage to intestinal epithelial tight junctions plays an important role in sepsis. Recently we found that Carbon Monoxide-Releasing Molecule-2 (CORM-2) is able to protect LPS-induced intestinal epithelial tight junction damage and in this study we will investigate if CORM-2 could protect intestinal epithelial tight junctions in the rat cecal ligation and puncture (CLP) model. Materials and Methods The CLP model was generated using male Sprague-Dawley (SD) rats according to standard procedure and treated with CORM-2 or inactive CORM-2 (iCORM-2), 8 mg/kg, i.v. immediately after CLP induction and euthanized after 24h or 72h (for mortality rate only). Morphological changes were investigated using both transmission electron and confocal microscopy. The levels of important TJ proteins and phosphorylation of myosin light chain (MLC) were examined using Western blotting. Cytokines, IL-1β and TNF-α were measured using ELISA kits. The overall intestinal epithelial permeability was evaluated using FD-4 as a marker. Results CORM-2, but not iCORM-2, significantly reduced sepsis-induced damage of intestinal mucosa (including TJ disruption), TJ protein reduction (including zonula occludens-l (ZO-1), claudin-1 and occludin), MLC phosphorylation and proinflammatory cytokine release. The overall outcomes showed that CORM-2 suppressed sepsis-induced intestinal epithelial permeability changes and reduced mortality rate of those septic rats. Conclusions Our data strongly suggest that CORM-2 could be a potential therapeutic reagent for sepsis by suppressing inflammation, restoring intestinal epithelial barrier and reducing mortality. PMID:26720630

  6. Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease

    PubMed Central

    Zeissig, S; Bürgel, N; Günzel, D; Richter, J; Mankertz, J; Wahnschaffe, U; Kroesen, A J; Zeitz, M; Fromm, M; Schulzke, J‐D

    2007-01-01

    Background Epithelial barrier function is impaired in Crohn's disease. Aim To define the underlying cellular mechanisms with special attention to tight junctions. Methods Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn's disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling and 4′,6‐diamidino‐2‐phenylindole staining. Results Patients with active Crohn's disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore‐forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn's disease compared with active ulcerative colitis and was inducible by tumour necrosis factor α. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn's disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis

  7. CHOLESTEROL DEPLETION ALTERS DETERGENT-SPECIFIC SOLUBILITY PROFILES OF SELECTED TIGHT JUNCTION PROTEINS AND THE PHOSPHORYLATION OF OCCLUDIN

    PubMed Central

    Lynch, Robert D.; Francis, Stacy A.; McCarthy, Karin M.; Casas, Elizabeth; Thiele, Christoph; Schneeberger, Eveline E.

    2007-01-01

    Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH) depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [3H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin’s presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH. PMID:17574235

  8. Double gene deletion reveals the lack of cooperation between claudin 11 and claudin 14 tight junction proteins

    PubMed Central

    Elkouby-Naor, Liron; Abassi, Zaid; Lagziel, Ayala; Gow, Alexander; Ben-Yosef, Tamar

    2010-01-01

    Summary Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. Selectivity and specificity of TJ strands are determined by the type of claudins present. It is thus important to understand the cooperation between different claudins in various tissues. To study the possible cooperation between claudin 11 and claudin 14 we generated claudin11/claudin 14 double deficient mice. These mice exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits and male sterility. In the kidney we found that these two claudins have distinct and partially overlapping expression patterns. Claudin 11 is located in both the proximal and the distal convoluted tubules, while claudin 14 is located in both the thin descending and the thick ascending limbs of the loop of Henle, as well as in the proximal convoluted tubules. Although daily urinary excretion of Mg++, and to a lesser extent of Ca++, tended to be higher in claudin11/claudin 14 double mutants, these changes did not reach statistical significance comparing to wt animals. These findings suggest that under normal conditions co-deletion of claudin11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished will provide further insight into the complex interactions between claudin proteins in various physiological systems. PMID:18663477

  9. Multi-junction solar cell device

    DOEpatents

    Friedman, Daniel J.; Geisz, John F.

    2007-12-18

    A multi-junction solar cell device (10) is provided. The multi-junction solar cell device (10) comprises either two or three active solar cells connected in series in a monolithic structure. The multi-junction device (10) comprises a bottom active cell (20) having a single-crystal silicon substrate base and an emitter layer (23). The multi-junction device (10) further comprises one or two subsequent active cells each having a base layer (32) and an emitter layer (23) with interconnecting tunnel junctions between each active cell. At least one layer that forms each of the top and middle active cells is composed of a single-crystal III-V semiconductor alloy that is substantially lattice-matched to the silicon substrate (22). The polarity of the active p-n junction cells is either p-on-n or n-on-p. The present invention further includes a method for substantially lattice matching single-crystal III-V semiconductor layers with the silicon substrate (22) by including boron and/or nitrogen in the chemical structure of these layers.

  10. Lipopolysaccharide-Induced Middle Ear Inflammation Disrupts the cochlear Intra-Strial Fluid–Blood Barrier through Down-Regulation of Tight Junction Proteins

    PubMed Central

    Zhang, Jinhui; Chen, Songlin; Hou, Zhiqiang; Cai, Jing; Dong, Mingmin; Shi, Xiaorui

    2015-01-01

    Middle ear infection (or inflammation) is the most common pathological condition that causes fluid to accumulate in the middle ear, disrupting cochlear homeostasis. Lipopolysaccharide, a product of bacteriolysis, activates macrophages and causes release of inflammatory cytokines. Many studies have shown that lipopolysaccharides cause functional and structural changes in the inner ear similar to that of inflammation. However, it is specifically not known how lipopolysaccharides affect the blood-labyrinth barrier in the stria vascularis (intra-strial fluid–blood barrier), nor what the underlying mechanisms are. In this study, we used a cell culture-based in vitro model and animal-based in vivo model, combined with immunohistochemistry and a vascular leakage assay, to investigate lipopolysaccharide effects on the integrity of the mouse intra-strial fluid–blood barrier. Our results show lipopolysaccharide-induced local infection significantly affects intra-strial fluid–blood barrier component cells. Pericytes and perivascular-resident macrophage-like melanocytes are particularly affected, and the morphological and functional changes in these cells are accompanied by substantial changes in barrier integrity. Significant vascular leakage is found in the lipopolysaccharide treated-animals. Consistent with the findings from the in vivo animal model, the permeability of the endothelial cell monolayer to FITC-albumin was significantly higher in the lipopolysaccharide-treated monolayer than in an untreated endothelial cell monolayer. Further study has shown the lipopolysaccharide-induced inflammation to have a major effect on the expression of tight junctions in the blood barrier. Lipopolysaccharide was also shown to cause high frequency hearing loss, corroborated by previous reports from other laboratories. Our findings show lipopolysaccharide-evoked middle ear infection disrupts inner ear fluid balance, and its particular effects on the intra-strial fluid

  11. Lipopolysaccharide-induced middle ear inflammation disrupts the cochlear intra-strial fluid-blood barrier through down-regulation of tight junction proteins.

    PubMed

    Zhang, Jinhui; Chen, Songlin; Hou, Zhiqiang; Cai, Jing; Dong, Mingmin; Shi, Xiaorui

    2015-01-01

    Middle ear infection (or inflammation) is the most common pathological condition that causes fluid to accumulate in the middle ear, disrupting cochlear homeostasis. Lipopolysaccharide, a product of bacteriolysis, activates macrophages and causes release of inflammatory cytokines. Many studies have shown that lipopolysaccharides cause functional and structural changes in the inner ear similar to that of inflammation. However, it is specifically not known how lipopolysaccharides affect the blood-labyrinth barrier in the stria vascularis (intra-strial fluid-blood barrier), nor what the underlying mechanisms are. In this study, we used a cell culture-based in vitro model and animal-based in vivo model, combined with immunohistochemistry and a vascular leakage assay, to investigate lipopolysaccharide effects on the integrity of the mouse intra-strial fluid-blood barrier. Our results show lipopolysaccharide-induced local infection significantly affects intra-strial fluid-blood barrier component cells. Pericytes and perivascular-resident macrophage-like melanocytes are particularly affected, and the morphological and functional changes in these cells are accompanied by substantial changes in barrier integrity. Significant vascular leakage is found in the lipopolysaccharide treated-animals. Consistent with the findings from the in vivo animal model, the permeability of the endothelial cell monolayer to FITC-albumin was significantly higher in the lipopolysaccharide-treated monolayer than in an untreated endothelial cell monolayer. Further study has shown the lipopolysaccharide-induced inflammation to have a major effect on the expression of tight junctions in the blood barrier. Lipopolysaccharide was also shown to cause high frequency hearing loss, corroborated by previous reports from other laboratories. Our findings show lipopolysaccharide-evoked middle ear infection disrupts inner ear fluid balance, and its particular effects on the intra-strial fluid-blood barrier

  12. 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.

  13. Similar and Distinct Properties of MUPP1 and Patj, Two Homologous PDZ Domain-Containing Tight-Junction Proteins ▿ †

    PubMed Central

    Adachi, Makoto; Hamazaki, Yoko; Kobayashi, Yuka; Itoh, Masahiko; Tsukita, Sachiko; Furuse, Mikio; Tsukita, Shoichiro

    2009-01-01

    MUPP1 and Patj are both composed of an L27 domain and multiple PDZ domains (13 and 10 domains, respectively) and are localized to tight junctions (TJs) in epithelial cells. Although Patj is known to be responsible for the organization of TJs and epithelial polarity, characterization of MUPP1 is lacking. In this study, we found that MUPP1 and Patj share several binding partners, including JAM1, ZO-3, Pals1, Par6, and nectins (cell-cell adhesion molecules at adherens junctions). MUPP1 and Patj exhibited similar subcellular distributions, and the mechanisms with which they localize to TJs also appear to overlap. Despite these similarities, functional studies have revealed that Patj is indispensable for the establishment of TJs and epithelial polarization, whereas MUPP1 is not. Thus, although MUPP1 and Patj share several molecular properties, their functions are entirely different. We present evidence that the signaling mediated by Pals1, which has a higher affinity for Patj than for MUPP1 and is involved in the activation of the Par6-aPKC complex, is of principal importance for the function of Patj in epithelial cells. PMID:19255144

  14. Epithelial adhesive junctions

    PubMed Central

    Capaldo, Christopher T.; Farkas, Attila E.

    2014-01-01

    Epithelial adhesive cell-to-cell contacts contain large, plasma membrane-spanning multiprotein aggregates that perform vital structural and signaling functions. Three prominent adhesive contacts are the tight junction, adherens junction, and the desmosome. Each junction type has unique cellular functions and a complex molecular composition. In this review, we comment on recent and exciting advances in our understanding of junction composition and function. PMID:24592313

  15. Mild hypothermia alleviates brain oedema and blood-brain barrier disruption by attenuating tight junction and adherens junction breakdown in a swine model of cardiopulmonary resuscitation

    PubMed Central

    Li, Jiebin; Li, Chunsheng; Yuan, Wei; Wu, Junyuan; Li, Jie; Li, Zhenhua; Zhao, Yongzhen

    2017-01-01

    Mild hypothermia improves survival and neurological recovery after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). However, the mechanism underlying this phenomenon is not fully elucidated. The aim of this study was to determine whether mild hypothermia alleviates early blood–brain barrier (BBB) disruption. We investigated the effects of mild hypothermia on neurologic outcome, survival rate, brain water content, BBB permeability and changes in tight junctions (TJs) and adherens junctions (AJs) after CA and CPR. Pigs were subjected to 8 min of untreated ventricular fibrillation followed by CPR. Mild hypothermia (33°C) was intravascularly induced and maintained at this temperature for 12 h, followed by active rewarming. Mild hypothermia significantly reduced cortical water content, decreased BBB permeability and attenuated TJ ultrastructural and basement membrane breakdown in brain cortical microvessels. Mild hypothermia also attenuated the CPR-induced decreases in TJ (occludin, claudin-5, ZO-1) and AJ (VE-cadherin) protein and mRNA expression. Furthermore, mild hypothermia decreased the CA- and CPR-induced increases in matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression and increased angiogenin-1 (Ang-1) expression. Our findings suggest that mild hypothermia attenuates the CA- and resuscitation-induced early brain oedema and BBB disruption, and this improvement might be at least partially associated with attenuation of the breakdown of TJ and AJ, suppression of MMP-9 and VEGF expression, and upregulation of Ang-1 expression. PMID:28355299

  16. Resolvin D1 reduces deterioration of tight junction proteins by upregulating HO-1 in LPS-induced mice.

    PubMed

    Xie, Wanli; Wang, Huiqing; Wang, Lei; Yao, Chengye; Yuan, Ruixia; Wu, Qingping

    2013-09-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary permeability with high mortality. Resolvin D1 (RvD1), which has potent anti-inflammatory and pro-resolving activity, can attenuate pulmonary edema in the animal model of ALI. However, the mechanism underlying the protection of RvD1 on pulmonary edema is still unknown. Here we explore the effects and mechanism of RvD1 on the disruption of tight junction protein that results in the permeability edema in a model of lipopolysaccharide (LPS)-induced ALI. The severity of pulmonary edema was assessed by wet-to-dry rate and Evans blue infiltration; expressions of tight junction (TJ) proteins occludin and zona occludin-1 (ZO-1) were examined by immunofluorescence staining and western blot; mRNA in lung tissue was studied by real time-PCR; the TUNEL kit was performed for the detection of apoptosis of pulmonary barrier. Twenty-four hours after LPS inhalation by mice, wet-to-dry rate and Evans blue infiltration indicated that pretreatment with RvD1 relieved the pulmonary edema and pulmonary capillary permeability. Moreover, RvD1 attenuated the LPS-induced deterioration of TJ protein ZO-1 and occludin significantly. And we found that RvD1 increased heme oxygenase-1 (HO-1) expression contributed to the protection on the deterioration of TJs. In addition, we found that RvD1 could reduce pulmonary cellular apoptosis in LPS-induced mice. In conclusion, RvD1 possesses the ability that relieves the pulmonary edema and restores pulmonary capillary permeability and reduces disruption of TJs in LPS-induced ALI of mice, at least in part, by upregulating HO-1 expression.

  17. Chlorogenic Acid Decreases Intestinal Permeability and Increases Expression of Intestinal Tight Junction Proteins in Weaned Rats Challenged with LPS

    PubMed Central

    Ruan, Zheng; Liu, Shiqiang; Zhou, Yan; Mi, Shumei; Liu, Gang; Wu, Xin; Yao, Kang; Assaad, Houssein; Deng, Zeyuan; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2014-01-01

    Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21±1 d of age; 62.26±2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS. PMID:24887396

  18. Chlorogenic acid decreases intestinal permeability and increases expression of intestinal tight junction proteins in weaned rats challenged with LPS.

    PubMed

    Ruan, Zheng; Liu, Shiqiang; Zhou, Yan; Mi, Shumei; Liu, Gang; Wu, Xin; Yao, Kang; Assaad, Houssein; Deng, Zeyuan; Hou, Yongqing; Wu, Guoyao; Yin, Yulong

    2014-01-01

    Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA) could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS) challenge. Thirty-two weaned male Sprague Dawley rats (21 ± 1 d of age; 62.26 ± 2.73 g) were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA) supplemented group (orally 20 mg/kg and 50 mg/kg body). Dietary supplementation with CHA decreased (P<0.05) the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05) in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05) villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05) intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05) by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05) in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS.

  19. Efficient mucus permeation and tight junction opening by dissociable "mucus-inert" agent coated trimethyl chitosan nanoparticles for oral insulin delivery.

    PubMed

    Liu, Min; Zhang, Jian; Zhu, Xi; Shan, Wei; Li, Lian; Zhong, Jiaju; Zhang, Zhirong; Huang, Yuan

    2016-01-28

    Oral administration of protein drugs is greatly impeded by the lack of drug carriers that can efficiently overcome the absorption barriers of mucosa tissue, which consists of not only epithelium but also a blanket of mucus gel. We herein report a novel self-assembled nanoparticle (NP) platform for oral delivery of insulin by facilitating the efficient permeation through both of these two barriers. The NP possesses a core composed of insulin and trimethyl chitosan (TMC), and a dissociable "mucus-inert" hydrophilic coating of N-(2-hydroxypropyl) methacrylamide copolymer (pHPMA) derivative. The NPs exhibited free Brownian motion and excellent permeability in mucus, which enabled the access of the NP core to the epithelial cell surface underneath the mucus. Moreover, investigation of NP behavior showed that the pHPMA molecules started to dissociate as the NP permeates through mucus, and the TMC NP core was then exposed to facilitate transepithelial transport via paracellular pathway. The pHPMA coating significantly improved transepithelial transport of TMC-based NP and their ability to open tight junctions between the mucus-secreting epithelial cells. Moreover, in diabetic rats, pHPMA coated NPs generated a prominent hypoglycemic response following oral administration, and exhibited a relative bioavailability 2.8-fold higher than that of uncoated TMC-based NPs. Our study provided the evidence of using pHPMA as "mucus-inert" agent to enhance mucus permeation of TMC-based NPs, and validated a novel strategy to overcome the multiple absorption barriers using NP platform with dissociable hydrophilic coating and TMC-based core possessing tight junction-opening ability.

  20. Differential effects of hydrocortisone and TNFα on tight junction proteins in an in vitro model of the human blood–brain barrier

    PubMed Central

    Förster, Carola; Burek, Malgorzata; Romero, Ignacio A; Weksler, Babette; Couraud, Pierre-Olivier; Drenckhahn, Detlev

    2008-01-01

    Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood–brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE-cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration of glucocorticoids induced expression of the targets occludin 2.75 ± 0.04-fold and claudin-5 up to 2.32 ± 0.11-fold, which is likely to contribute to the more than threefold enhancement of transendothelial electrical resistance reflecting barrier tightness. Our analyses further provide direct evidence that the GC hydrocortisone prevents endothelial barrier breakdown in response to pro-inflammatory stimuli (TNFα administration), which could be demonstrated to be partly based on maintenance of occludin levels. Our studies strongly suggest stabilization of BBB function as a mode of GC action on a molecular level in the human brain vasculature. PMID:18258663

  1. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  2. A high-grain diet alters the omasal epithelial structure and expression of tight junction proteins in a goat model.

    PubMed

    Liu, Jun-Hua; Xu, Ting-Ting; Zhu, Wei-Yun; Mao, Sheng-Yong

    2014-07-01

    The omasal epithelial barrier plays important roles in maintaining nutrient absorption and immune homeostasis in ruminants. However, little information is currently available about the changes in omasal epithelial barrier function at the structural and molecular levels during feeding of a high-grain (HG) diet. Ten male goats were randomly assigned to two groups, fed either a hay diet (0% grain; n = 5) or HG diet (65% grain; n = 5). Changes in omasal epithelial structure and expression of tight junction (TJ) proteins were determined via electron microscopy and Western blot analysis. After 7 weeks on each diet, omasal contents in the HG group showed significantly lower pH (P <0.001) and significantly higher concentrations of free lipopolysaccharides (LPS; P = 0.001) than the hay group. The goats fed a HG diet showed profound alterations in omasal epithelial structure and TJ proteins, corresponding to depression of thickness of total epithelia, stratum granulosum, and the sum of the stratum spinosum and stratum basale, marked epithelial cellular damage, erosion of intercellular junctions and down-regulation in expression of the TJ proteins, claudin-4 and occludin. The study demonstrates that feeding a HG diet is associated with omasal epithelial cellular damage and changes in expression of TJ proteins. These research findings provide an insight into the possible significance of diet on the omasal epithelial barrier in ruminants.

  3. Studies of silicon PN junction solar cells

    NASA Technical Reports Server (NTRS)

    Lindholm, F. A.

    1975-01-01

    Silicon pn junction solar cells made with low-resistivity substrates show poorer performance than traditional theory predicts. The purpose of this research was to identify and characterize the physical mechanisms responsible for the discrepancy. Attention was concentrated on the open circuit voltage in shallow junction cells of 0.1 ohm-cm substrate resistivity. A number of possible mechanisms that can occur in silicon devices were considered. Two mechanisms which are likely to be of main importance in explaining the observed low values of open-circuit voltage were found: (1) recombination losses associated with defects introduced during junction formation, and (2) inhomogeneity of defects and impurities across the area of the cell. To explore these theoretical anticipations, various diode test structures were designed and fabricated and measurement configurations for characterizing the defect properties and the areal inhomogeneity were constructed.

  4. 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

  5. Tight Junction Proteins and Oxidative Stress in Heavy Metals-Induced Nephrotoxicity

    PubMed Central

    Reyes, José L.; Molina-Jijón, Eduardo; Rodríguez-Muñoz, Rafael; Bautista-García, Pablo; Debray-García, Yazmin; Namorado, María del Carmen

    2013-01-01

    Kidney is a target organ for heavy metals. They accumulate in several segments of the nephron and cause profound alterations in morphology and function. Acute intoxication frequently causes acute renal failure. The effects of chronic exposure have not been fully disclosed. In recent years increasing awareness of the consequences of their presence in the kidney has evolved. In this review we focus on the alterations induced by heavy metals on the intercellular junctions of the kidney. We describe that in addition to the proximal tubule, which has been recognized as the main site of accumulation and injury, other segments of the nephron, such as glomeruli, vessels, and distal nephron, show also deleterious effects. We also emphasize the participation of oxidative stress as a relevant component of the renal damage induced by heavy metals and the beneficial effect that some antioxidant drugs, such as vitamin A (all-trans-retinoic acid) and vitamin E (α-tocopherol), depict on the morphological and functional alterations induced by heavy metals. PMID:23710457

  6. Tight junction proteins and oxidative stress in heavy metals-induced nephrotoxicity.

    PubMed

    Reyes, José L; Molina-Jijón, Eduardo; Rodríguez-Muñoz, Rafael; Bautista-García, Pablo; Debray-García, Yazmin; Namorado, María Del Carmen

    2013-01-01

    Kidney is a target organ for heavy metals. They accumulate in several segments of the nephron and cause profound alterations in morphology and function. Acute intoxication frequently causes acute renal failure. The effects of chronic exposure have not been fully disclosed. In recent years increasing awareness of the consequences of their presence in the kidney has evolved. In this review we focus on the alterations induced by heavy metals on the intercellular junctions of the kidney. We describe that in addition to the proximal tubule, which has been recognized as the main site of accumulation and injury, other segments of the nephron, such as glomeruli, vessels, and distal nephron, show also deleterious effects. We also emphasize the participation of oxidative stress as a relevant component of the renal damage induced by heavy metals and the beneficial effect that some antioxidant drugs, such as vitamin A (all-trans-retinoic acid) and vitamin E ( α -tocopherol), depict on the morphological and functional alterations induced by heavy metals.

  7. Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor

    PubMed Central

    Verma, Saguna; Kumar, Mukesh; Gurjav, Ulziijargal; Lum, Stephanie; Nerurkar, Vivek R.

    2011-01-01

    Though compromised blood-brain barrier (BBB) is a pathological hallmark of WNV- associated neurological sequelae, underlying mechanisms are unclear. We characterized the expression of matrix metalloproteinases (MMP) in WNV-infected human brain-microvascular endothelial cells (HBMVE) and -cortical astrocytes (HBCA), components of BBB and their role in BBB disruption. Expression of multiple MMPs was significantly induced in WNV-infected HBCA cells. Naïve HBMVE cells incubated with the supernatant from WNV-infected HBCA cells demonstrated loss of tight junction proteins, which was rescued in the presence of MMP inhibitor, GM6001. Further, supernatant from WNV-infected HBCA cells compromised the in-vitro BBB models integrity. Our data suggests astrocytes as one of the sources of MMP in the brain, which mediates BBB disruption allowing unrestricted entry of immune cells into the brain, thereby contributing to WNV-neuropathogenesis. Because of the unavailability of WNV antivirals and vaccines, use of MMP inhibitors as an adjunct therapy to ameliorate WNV disease progression is warranted. PMID:19922973

  8. Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: Relevance in barrier integrity and tight junction formation and complexity.

    PubMed

    Freese, Christian; Hanada, Sanshiro; Fallier-Becker, Petra; Kirkpatrick, C James; Unger, Ronald E

    2017-05-01

    We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm(2) was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.

  9. EFA6, exchange factor for ARF6, regulates the actin cytoskeleton and associated tight junction in response to E-cadherin engagement.

    PubMed

    Luton, Frédéric; Klein, Stéphanie; Chauvin, Jean-Paul; Le Bivic, André; Bourgoin, Sylvain; Franco, Michel; Chardin, Pierre

    2004-03-01

    We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin-mediated cell adhesion, EFA6 is recruited to a Triton X-100-insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability.

  10. Mechanism of IFN-γ-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane

    PubMed Central

    Utech, Markus; Ivanov, Andrei I.; Samarin, Stanislav N.; Bruewer, Matthias; Turner, Jerrold R.; Mrsny, Randall J.; Parkos, Charles A.; Nusrat, Asma

    2005-01-01

    Disruption of epithelial barrier by proinflammatory cytokines such as IFN-γ represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-γ increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-γ-induced endocytosis of epithelial TJ proteins. IFN-γ treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-γ dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-γ exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-γ treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-γ induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs. PMID:16055505

  11. Nitric Oxide and Airway Epithelial Barrier Function: Regulation of Tight Junction Proteins and Epithelial Permeability

    PubMed Central

    Olson, Nels; Greul, Anne-Katrin; Hristova, Milena; Bove, Peter F.; Kasahara, David I.; van der Vliet, Albert

    2008-01-01

    Acute airway inflammation is associated with enhanced production of nitric oxide (NO•) and altered airway epithelial barrier function, suggesting a role of NO• or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o- cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2-/- mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H2O2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity. PMID:19100237

  12. Disruption of Tight Junctions by Cellulose Sulfate Facilitates HIV Infection: Model of Microbicide Safety

    PubMed Central

    Mesquita, Pedro M. M.; Cheshenko, Natalia; Wilson, Sarah S.; Mhatre, Mohak; Guzman, Esmeralda; Fakioglu, Esra; Keller, Marla J.; Herold, Betsy C.

    2010-01-01

    Background The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. Methods Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immuodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. Results Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor κB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. Conclusions Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes. PMID:19586414

  13. Impaired Tight Junctions in Atopic Dermatitis Skin and in a Skin-Equivalent Model Treated with Interleukin-17

    PubMed Central

    Yuki, Takuo; Tobiishi, Megumi; Kusaka-Kikushima, Ayumi; Ota, Yukiko; Tokura, Yoshiki

    2016-01-01

    Tight junction (TJ) dysfunction in the stratum granulosum leads to aberrant barrier function of the stratum corneum (SC) in the epidermis. However, it is unclear whether TJs are perturbed in atopic dermatitis (AD), a representative aberrant SC-related skin disease, and whether some factors related to AD pathogenesis induce TJ dysfunction. To address these issues, we investigated the alterations of TJs in AD skin and the effects of Th2 and Th17 cytokines on TJs in a skin-equivalent model. The levels of TJ proteins were determined in the epidermis of nonlesional and lesional skin sites of AD. Western blot and immunohistochemical analyses revealed that the levels of zonula occludens 1 were decreased in the nonlesional sites of AD, and the levels of zonula occludens 1 and claudin-1 were decreased in the lesional sites relative to the levels in skin from healthy subjects. Next, we examined the effects of interleukin (IL)-4, tumor necrosis factor-α, IL-17, and IL-22 on the TJ barrier in a skin-equivalent model. Only IL-17 impaired the TJ barrier. Furthermore, we observed a defect in filaggrin monomer degradation in the IL-17–treated skin model. Thus, TJs are dysfunctional in AD, at least partly, due to the effect of IL-17, which may result in an aberrant SC barrier. PMID:27588419

  14. Quantitative atlas of blood-brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset.

    PubMed

    Hoshi, Yutaro; Uchida, Yasuo; Tachikawa, Masanori; Inoue, Takashi; Ohtsuki, Sumio; Terasaki, Tetsuya

    2013-09-01

    The purpose of this study was to determine the protein amounts of blood-brain barrier (BBB) permeability-related transporters, receptors, and tight junction proteins in Sprague Dawley and Wistar rats and common marmoset, and also to investigate inter-species and inter-strain differences across rodents and primates. Quantification of target proteins in isolated brain capillaries was conducted by liquid chromatography-tandem mass spectrometry-based quantitative targeted absolute proteomics, with in silico peptide selection. Most target proteins showed inter-rodent, inter-primate species, and inter-rat strain differences of less than 2-fold. Comparison of rat and human BBB showed that P-glycoprotein, multidrug resistance-associated protein 4, monocarboxylate transporter 1, l-type amino acid transporter, and organic anion transporter 3 exhibited differences of more than two-fold in protein abundance, whereas the amounts of breast cancer resistance protein, glucose transporter 1, and insulin receptor were similar in rat and human. In contrast, the differences between marmoset and human BBB were less than 2-fold for almost all measured proteins. Thus, the molecular basis of BBB functions may be similar in marmoset and human, whereas that of rats shows significant differences. The marmoset may be a good model to access in vivo human BBB permeability characteristics, as an alternative to rat and macaque monkey.

  15. Optimization of an immunohistochemical method to assess distribution of tight junction proteins in canine epidermis and adnexae.

    PubMed

    Roussel, A J J; Knol, A C; Bourdeau, P J; Bruet, V

    2014-01-01

    Epidermal tight junctions (TJs) have been well characterized in human medicine. Abnormality of these structures is involved in skin diseases such as atopic dermatitis. There is little information about the expression and distribution of TJ proteins in the canine skin. The aim of this study was to develop an optimal immunohistochemical method for assessment of the expression of TJ proteins in the skin of healthy dogs. Formalin-fixed and paraffin wax-embedded skin biopsy samples from healthy human and canine patients were used. Canine skin samples were from the inguinal region and the nasal planum. Immunohistochemistry was used to study the expression of zonula occludens-1 (ZO-1), occludin and claudin-1, -4 and -7. Heat-induced antigen retrieval with EDTA (pH 9.0) yielded the best labelling of TJ proteins. ZO-1 and occludin were expressed in the cytoplasm and along the keratinocyte membrane, while claudin-1 and -4 were mainly membrane in distribution. ZO-1, occludin and claudin-1 were detected in all epidermal layers with the exception of the stratum corneum, while claudin-4 expression was restricted to the stratum granulosum. Expression of claudin-7 was difficult to evaluate. There was no difference in labelling pattern between inguinal and nasal planum skin.

  16. Electroacupuncture Treatment Improves Neurological Function Associated with Regulation of Tight Junction Proteins in Rats with Cerebral Ischemia Reperfusion Injury

    PubMed Central

    Zhang, Ya-min; Xu, Hong; Sun, Hua; Chen, Su-hui; Wang, Fu-ming

    2014-01-01

    Strategies to develop effective neuroprotective therapy to reduce brain damage and related behavioral deficits in stroke patients are of great significance. Electroacupuncture (EA), which derives from traditional Chinese medicine, may be effective as a complementary and alternative method for promoting recovery of neurological function and quality of life. Adult Sprague-Dawley rats were randomly divided into 3 groups: (1) sham, (2) middle cerebral artery occlusion (MCAO) model groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion, and (3) EA groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion. EA groups received EA therapy by needling at GV20 and left ST36. The results show that EA therapy improved the neurological function and reduced infarct volume, confirmed by modified neurological severity scores and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that EA upregulated the expression of tight junction (TJ) claudin-5, occludin, and zonula occluding-1 from 1 to 7 d after reperfusion. Our findings suggest that EA reduces brain damage and related behavioral deficits via upregulation of the TJ proteins. PMID:25009574

  17. Electroacupuncture treatment improves neurological function associated with regulation of tight junction proteins in rats with cerebral ischemia reperfusion injury.

    PubMed

    Zhang, Ya-Min; Xu, Hong; Sun, Hua; Chen, Su-Hui; Wang, Fu-Ming

    2014-01-01

    Strategies to develop effective neuroprotective therapy to reduce brain damage and related behavioral deficits in stroke patients are of great significance. Electroacupuncture (EA), which derives from traditional Chinese medicine, may be effective as a complementary and alternative method for promoting recovery of neurological function and quality of life. Adult Sprague-Dawley rats were randomly divided into 3 groups: (1) sham, (2) middle cerebral artery occlusion (MCAO) model groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion, and (3) EA groups of 2 h MCAO followed by 1, 3, 5, or 7 d of reperfusion. EA groups received EA therapy by needling at GV20 and left ST36. The results show that EA therapy improved the neurological function and reduced infarct volume, confirmed by modified neurological severity scores and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that EA upregulated the expression of tight junction (TJ) claudin-5, occludin, and zonula occluding-1 from 1 to 7 d after reperfusion. Our findings suggest that EA reduces brain damage and related behavioral deficits via upregulation of the TJ proteins.

  18. Claudin-8 and -27 tight junction proteins in puffer fish Tetraodon nigroviridis acclimated to freshwater and seawater.

    PubMed

    Bagherie-Lachidan, Mazdak; Wright, Stephen I; Kelly, Scott P

    2009-05-01

    Genes encoding for claudin-8 and -27 tight junction proteins in the euryhaline puffer fish (Tetraodon nigroviridis) were identified using its recently sequenced genome. Phylogenetic analysis indicated that multiple genes encoding for claudin-8 proteins (designated Tncldn8a, Tncldn8b, Tncldn8c and Tncldn8d) arose by tandem gene duplication. In contrast, both tandem and whole genome duplication events appear to have generated genes encoding for claudin-27 proteins (designated Tncldn27a, Tncldn27b, Tncldn27c and Tncldn27d). Tncldn8 and Tncldn27 mRNA were widely distributed in Tetraodon, suggesting involvement in various physiological processes. All Tncldn8 and Tncldn27 genes were expressed in gill and skin tissue (i.e., epithelia exposed directly to the external environment). A potential role for claudin-8 and -27 proteins in the regulation of hydromineral balance in Tetraodon was investigated by examining alterations in mRNA abundance in select ionoregulatory tissue of fish acclimated to freshwater (FW) and seawater (SW). In FW or SW, Tetraodon exhibited alterations in Na(+)-K(+)-ATPase activity (a correlate of transcellular transport) typical of a euryhaline teleost fish. Simultaneously, tissue and gene specific alterations in Tncldn8 and Tncldn27 transcript abundance occurred. These data provide some insight into the duplication history of cldn8 and cldn27 genes in fishes and suggest a possible role for claudin-8 and -27 proteins in the osmoregulatory strategies of euryhaline teleosts.

  19. S0 Tight Loop Studies on ICHIRO 9-Cell Cavities

    SciTech Connect

    Furuta, Fumio; Konomi, T.; Saito, Kenji; Bice, Damon; Crawford, Anthony C.; Geng, Rongli

    2009-11-01

    We have continued high gradient R&D of ICHIRO 9-cell cavities at KEK. ICHIRO 9-cell cavity #5 (I9#5) that has no end groups on beam tube to focus on high gradient sent to Jlab as S0 tight loop study. Surface treatments and vertical test were repeated 3 times at Jlab, and then I9#5 sent back to KEK. We also repeated surface treatments and test at KEK. Maximum gradients were 36.5MV/m at Jlab, and 33.7MV/m at KEK so far. Now we are struggling with the puzzle why the results of singles do not work well on 9-cell cavities.

  20. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

    Membrane permeability of an epithelial cell junction (Drosophila salivary gland) was examined with intracellular microelectrodes and with fluorescent tracers. In contrast to the non-junctional cell membrane surface, which has a low permeability to ions (10-4 mho/cm2), the junctional membrane surface is highly permeable. In fact, it introduces no substantial restriction to ion flow beyond that in the cytoplasm; the resistance through a chain of cells (150 Ω cm) is only slightly greater than in extruded cytoplasm (100 Ω cm). The diffusion resistance along the intercellular space to the exterior, on the other hand, is very high. Here, there exists an ion barrier of, at least, 104Ω cm2. As a result, small ions and fluorescein move rather freely from one cell to the next, but do not leak appreciably through the intercellular space to the exterior. The organ here, rather than the single cell, appears to be the unit of ion environment. The possible underlying structural aspects are discussed. PMID:14206423

  1. Tight junction protein ZO-1 controls organic cation/carnitine transporter OCTN2 (SLC22A5) in a protein kinase C-dependent way.

    PubMed

    Jurkiewicz, Dominika; Michalec, Katarzyna; Skowronek, Krzysztof; Nałęcz, Katarzyna A

    2017-05-01

    OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome. We showed previously that activation of protein kinase C (PKC) in rat astrocytes regulates Octn2 surface presence and activity. Regulation of a wild type Octn2 and its deletion mutant without a PDZ binding motif were studied in heterologous expression system in HEK293 cells. Plasma membrane presence of overexpressed Octn2 did not depend on either PKC activation or presence of PDZ-binding motif, anyhow, as assayed in proximity ligation assay, the truncation of PDZ binding motif resulted in a strongly diminished Octn2/ZO-1 interaction and in a decreased transporter activity. The same effects on Octn2 activity were detected upon PKC activation, what correlated with ZO-1 phosphorylation. It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in ΔPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity.

  2. Claudin tight junction proteins in rainbow trout (Oncorhynchus mykiss) skin: Spatial response to elevated cortisol levels.

    PubMed

    Gauberg, Julia; Kolosov, Dennis; Kelly, Scott P

    2017-01-01

    This study examined regional distribution and corticosteroid-induced alterations of claudin (cldn) transcript abundance in teleost fish skin. Regional comparison of mRNA encoding 20 Cldns indicated that 12 exhibit differences in abundance along the dorsoventral axis of skin. However, relative abundance of cldns (i.e. most to least abundant) remained similar in different skin regions. Several cldns appear to be present in the epidermis and dermal vasculature whereas others are present only in the epidermis. Increased circulating cortisol levels significantly altered mRNA abundance of 10 cldns in a region specific manner, as well as corticosteroid receptors and 11β-hydroxysteroid dehydrogenase (type 2). Epidermis and epidermal mucous cell morphometrics also altered in response to cortisol, exhibiting changes that appear to enhance skin barrier properties. Taken together, data provide a first look at spatial variation in the molecular physiology of the teleost fish integument TJ complex and region-specific sensitivity to an endocrine factor.

  3. Structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus.

    PubMed

    Jung, Kwonil; Eyerly, Bryan; Annamalai, Thavamathi; Lu, Zhongyan; Saif, Linda J

    2015-06-12

    Integrity of the intestinal epithelium is critical for proper functioning of the barrier that regulates absorption of water and restricts uptake of luminal bacteria. It is maintained mainly by tight junctions (TJs) and adherens junctions (AJs). We conducted immunofluorescence (IF) staining for in situ identification of zonula occludin (ZO)-1 proteins for TJ and E-Cadherin proteins for AJ in the small and large intestinal villous and crypt epithelium of nursing pigs infected with porcine epidemic diarrhea virus (PEDV). Twenty 9-day-old piglets [PEDV-infected (n=9) and Mock (n=11)] from PEDV seronegative sows, were orally inoculated [8.9 log₁₀ genomic equivalents/pig] with PEDV PC21A strain or mock. At post-inoculation days (PIDs) 1-5, infected pigs showed severe watery diarrhea and/or vomiting and severe atrophic enteritis. By immunohistochemistry, PEDV antigens were evident in enterocytes lining the villous epithelium. At PIDs 1-5, PEDV-infected pigs exhibited mildly to extensively disorganized, irregular distribution and reduced expression of ZO-1 or E-Cadherin in villous, but not crypt epithelial cells of the jejunum and ileum, but not in the large intestine, when compared to the negative controls. The structural destruction and disorganization of TJ and AJ were extensive in PEDV-infected pigs at PIDs 1-3, but then appeared to reversibly recover at PID 5, as evident by increased numbers of ZO-1-positive epithelial cells and markedly improved appearance of E-Cadherin-positive villous epithelium. Our results suggest a possible involvement of structurally impaired TJ and AJ in the pathogenesis of PEDV, potentially leading to secondary bacterial infections.

  4. Hyperbaric oxygen preconditioning ameliorates blood-brain barrier damage induced by hypoxia through modulation of tight junction proteins in an in vitro model

    PubMed Central

    Hao, Lei; Guo, Xiuming; Zou, Can; Zhou, Huchuan; Tian, Hong; Zhang, Yubo; Song, Chuan; Liu, Lei

    2016-01-01

    Aim To explore the effects of hyperbaric oxygen preconditioning (HBOP) on the permeability of blood-brain barrier (BBB) and expression of tight junction proteins under hypoxic conditions in vitro. Methods A BBB in vitro model was constructed using the hCMEC/D3 cell line and used when its trans-endothelial electrical resistance (TEER) reached 80-120 Ω · cm2 (tested by Millicell-Electrical Resistance System). The cells were randomly divided into the control group cultured under normal conditions, the group cultured under hypoxic conditions (2%O2) for 24 h (hypoxia group), and the group first subjected to HBOP for 2 h and then to hypoxia (HBOP group). Occludin and ZO-1 expression were analyzed by immunofluorescence assay. Results Normal hCMEC/D3 was spindle-shaped and tightly integrated. TEER was significantly reduced in the hypoxia (P = 0.001) and HBOP group (P = 0.014) compared to control group, with a greater decrease in the hypoxia group. Occludin membranous expression was significantly decreased in the hypoxia group (P = 0.001) compared to the control group, but there was no change in the HBOP group. ZO-1 membranous expression was significantly decreased (P = 0.002) and cytoplasmic expression was significantly increased (P = 0.001) in the hypoxia group compared to the control group, although overall expression levels did not change. In the HBOP group, there was no significant change in ZO-1 expression compared to the control group. Conclusion Hyperbaric oxygen preconditioning protected the integrity of BBB in an in vitro model through modulation of occludin and ZO-1 expression under hypoxic conditions. PMID:26935614

  5. Plasmon Enhanced Hetero-Junction Solar Cell

    NASA Astrophysics Data System (ADS)

    Long, Gen; Ching, Levine; Sadoqi, Mostafa; Xu, Huizhong

    2015-03-01

    Here we report a systematic study of plasmon-enhanced hetero-junction solar cells made of colloidal quantum dots (PbS) and nanowires (ZnO), with/without metal nanoparticles (Au). The structure of solar cell devices was characterized by AFM, SEM and profilometer, etc. The power conversion efficiencies of solar cell devices were characterized by solar simulator (OAI TriSOL, AM1.5G Class AAA). The enhancement in the photocurrent due to introduction of metal nanoparticles was obvious. We believe this is due to the plasmonic effect from the metal nanoparticles. The correlation between surface roughness, film uniformity and device performance was also studied.

  6. Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins.

    PubMed

    Xiao, Lan; Rao, Jaladanki N; Cao, Shan; Liu, Lan; Chung, Hee Kyoung; Zhang, Yun; Zhang, Jennifer; Liu, Yulan; Gorospe, Myriam; Wang, Jian-Ying

    2016-02-15

    Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs.

  7. Different Changes in the Expression of Multiple Kinds of Tight-Junction Proteins during Ischemia-Reperfusion Injury of the Rat Ileum

    PubMed Central

    Inoue, Kaori; Oyamada, Masahito; Mitsufuji, Shoji; Okanoue, Takeshi; Takamatsu, Tetsuro

    2006-01-01

    Dysfunction of tight junctions (TJs), located at the most apical part of the intestinal epithelium, is believed to result in various complications in intestinal disease. However, the behaviors of multiple kinds of TJ proteins during ischemia-reperfusion injury are not understood in detail. To determine changes in expression and localization of TJ proteins during intestinal-barrier recovery, we induced intestinal ischemia-reperfusion injury in rats, measured mucosa-to-blood permeability of fluorescein isothiocyanate-dextran-4 kDa, and compared it with spatiotemporal changes of ZO-1, occludin, and claudin-1, -2, -3, -4, and -5 by immunoconfocal microscopy. At 1 hour post-reperfusion, villi were denuded and intestinal-barrier function was lost. From 6 to 24 hours post-reperfusion, villous epithelium was restored by cell migration, and barrier function together with reticular pattern expression of ZO-1, occludin, and claudin-1, -3, and -5, recovered time-dependently. To the contrary, after ischemia-reperfusion injury, the localized expression of claudin-2 and claudin-4 observed in the non-treated control was lost and replaced with broader expression from crypts to villi with increased basolateral claudin-4 expression in epithelial cells. These data demonstrated that recovery of intestinal barrier function is associated with expression of ZO-1, occludin, and claudin-1, -3, and -5, whereas claudin-2 and claudin-4 show unique changes in expression and localization. PMID:17375208

  8. Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins

    PubMed Central

    Xiao, Lan; Rao, Jaladanki N.; Cao, Shan; Liu, Lan; Chung, Hee Kyoung; Zhang, Yun; Zhang, Jennifer; Liu, Yulan; Gorospe, Myriam; Wang, Jian-Ying

    2016-01-01

    Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs. PMID:26680741

  9. Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.

    PubMed

    Nighot, Prashant K; Leung, Lana; Ma, Thomas Y

    2017-03-01

    Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2(CLCN2)) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2(CLCN2)) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2(pEZ) cells. ClC-2 over-expression (Caco-2(CLCN2)) increased occludin protein level compared to control Caco-2(pEZ) cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2(CLCN2) cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2(CLCN2) cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

  10. Changes in integrity of the gill during histidine deficiency or excess due to depression of cellular anti-oxidative ability, induction of apoptosis, inflammation and impair of cell-cell tight junctions related to Nrf2, TOR and NF-κB signaling in fish.

    PubMed

    Jiang, Wei-Dan; Feng, Lin; Qu, Biao; Wu, Pei; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2016-09-01

    integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, apoptosis, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gills.

  11. F-actin binding protein, anillin, regulates integrity of intercellular junctions in human epithelial cells

    PubMed Central

    Feygin, Alex; Ivanov, Andrei I.

    2015-01-01

    Tight junctions (TJ) and adherens junctions (AJ) are key morphological features of differentiated epithelial cells that regulate the integrity and permeability of tissue barriers. Structure and remodeling of epithelial junctions depends on their association with the underlying actomyosin cytoskeleton. Anillin is a unique scaffolding protein interacting with different cytoskeletal components, including actin filaments and myosin motors. Its role in the regulation of mammalian epithelial junctions remains unexplored. Downregulation of anillin expression in human prostate, colonic, and lung epithelial cells triggered AJ and TJ disassembly without altering the expression of junctional proteins. This junctional disassembly was accompanied by dramatic disorganization of the perijunctional actomyosin belt; while the general architecture of the actin cytoskeleton, and activation status of non-muscle myosin II, remained unchanged. Furthermore, loss of anillin disrupted the adducin-spectrin membrane skeleton at the areas of cell-cell contact, selectively decreased γ-adducin expression, and induced cytoplasmic aggregation of αII-spectrin. Anillin knockdown activated c-Jun N-terminal kinase (JNK), and JNK inhibition restored AJ and TJ integrity and cytoskeletal organization in anillin-depleted cells. These findings suggest a novel role for anillin in regulating intercellular adhesion in model human epithelia by mechanisms involving the suppression of JNK activity and controlling the assembly of the perijunctional cytoskeleton. PMID:25809162

  12. Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs.

    PubMed

    Metzler-Zebeli, Barbara U; Mann, Evelyne; Ertl, Reinhard; Schmitz-Esser, Stephan; Wagner, Martin; Klein, Dieter; Ritzmann, Mathias; Zebeli, Qendrim

    2015-04-14

    Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearman's correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearman's correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs.

  13. Decreased interaction between ZO-1 and occludin is involved in alteration of tight junctions in transplanted epiphora submandibular glands.

    PubMed

    Ding, Chong; Cong, Xin; Zhang, Xue-Ming; Li, Sheng-Lin; Wu, Li-Ling; Yu, Guang-Yan

    2017-03-22

    Tight junctions (TJs) in salivary epithelium play an important role in regulating saliva secretion. Autologous transplantation of submandibular glands (SMGs) is an effective method to treat severe dry eye syndrome. However, epiphora occurs in some patients 6 months after transplantation. We previously found that the acinar TJs are enlarged in rabbit SMGs after long-term transplantation, but the exact TJ components involved in the epiphora are still unknown. Here, we found that the mRNA and protein expression of ZO-1 and occludin were increased in the transplanted SMGs obtained from epiphora patients, while other TJs were unchanged. The intensity of ZO-1 and occludin at the apicolateral membranes as well as occludin in the cytoplasm were increased in epiphora SMGs, but the interaction between ZO-1 and occludin was decreased as evidenced by both co-immunoprecipitation assay and co-immunofluorescence staining. Mechanically, the expression of casein kinase 2α (CK2α) and CK2β, which was reported to affect occludin modification and the interaction of occludin with ZO-1 in previous literatures, were increased in epiphora glands. Moreover, activation of muscarinic acetylcholine receptor (mAChR) by carbachol directly decreased the interaction between ZO-1 and occludin and increased the acinar TJ width in the freshly isolated human SMGs, whereas these effects were abolished by pretreatment with CK2 inhibitor. Taken together, our findings suggest that decreased interaction between ZO-1 and occludin might contribute to the epiphora occurred in the transplanted SMGs, and mAChR together with the intracellular molecule CK2 might be responsible for the alteration of TJs in epiphora glands.

  14. Fetal DNA hypermethylation in tight junction pathway is associated with neural tube defects: A genome-wide DNA methylation analysis.

    PubMed

    Wang, Linlin; Lin, Shanshan; Zhang, Ji; Tian, Tian; Jin, Lei; Ren, Aiguo

    2017-02-01

    Neural tube defects (NTDs) are a spectrum of severe congenital malformations of fusion failure of the neural tube during early embryogenesis. Evidence on aberrant DNA methylation in NTD development remains scarce, especially when exposure to environmental pollutant is taken into consideration. DNA methylation profiling was quantified using the Infinium HumanMethylation450 array in neural tissues from 10 NTD cases and 8 non-malformed controls (stage 1). Subsequent validation was performed using a Sequenom MassARRAY system in neural tissues from 20 NTD cases and 20 non-malformed controls (stage 2). Correlation analysis of differentially methylated CpG sites in fetal neural tissues and polycyclic aromatic hydrocarbons concentrations in fetal neural tissues and maternal serum was conducted. Differentially methylated CpG sites of neural tissues were further validated in fetal mice with NTDs induced by benzo(a)pyrene given to pregnant mice. Differentially hypermethylated CpG sites in neural tissues from 17 genes and 6 pathways were identified in stage 1. Subsequently, differentially hypermethylated CpG sites in neural tissues from 6 genes (BDKRB2, CTNNA1, CYFIP2, MMP7, MYH2, and TIAM2) were confirmed in stage 2. Correlation analysis showed that methylated CpG sites in CTNNA1 and MYH2 from NTD cases were positively correlated to polycyclic aromatic hydrocarbon level in fetal neural tissues and maternal serum. The correlation was confirmed in NTD-affected fetal mice that were exposed to benzo(a)pyrene in utero. In conclusion, hypermethylation of the CTNNA1 and MYH2 genes in tight junction pathway is associated with the risk for NTDs, and the DNA methylation aberration may be caused by exposure to benzo(a)pyrene.

  15. Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress.

    PubMed

    McCaffrey, Gwen; Willis, Colin L; Staatz, William D; Nametz, Nicole; Quigley, Carolyn A; Hom, Sharon; Lochhead, Jeffrey J; Davis, Thomas P

    2009-07-01

    Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood-brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O(2), 60 min), Hx (6% O(2), 60 min), or hypoxia/reoxygenation (H/R, 6% O(2), 60 min followed by 21% O(2), 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.

  16. Excess junction current of silicon solar cells

    NASA Technical Reports Server (NTRS)

    Wang, E. Y.; Legge, R. N.; Christidis, N.

    1973-01-01

    The current-voltage characteristics of n(plus)-p silicon solar cells with 0.1, 1.0, 2.0, and 10 ohm-cm p-type base materials have been examined in detail. In addition to the usual I-V measurements, we have studied the temperature dependence of the slope of the I-V curve at the origin by the lock-in technique. The excess junction current coefficient (Iq) deduced from the slope at the origin depends on the square root of the intrinsic carrier concentration. The Iq obtained from the I-V curve fitting over the entire forward bias region at various temperatures shows the same temperature dependence. This result, in addition to the presence of an aging effect, suggest that the surface channel effect is the dominant cause of the excess junction current.

  17. Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus.

    PubMed

    Yoo, Dae Young; Yim, Hee Sun; Jung, Hyo Young; Nam, Sung Min; Kim, Jong Whi; Choi, Jung Hoon; Seong, Je Kyung; Yoon, Yeo Sung; Kim, Dae Won; Hwang, In Koo

    2016-07-01

    In the present study, we investigated the effects of type 2 diabetes-induced hyperglycemia on the integrity of the blood-brain barrier and tight junction markers in the rat hippocampus. Forty-week-old diabetic (Zucker diabetic fatty, ZDF) rats and littermate control (Zucker lean control, ZLC) rats were used in this study. We evaluated the integrity of the blood-brain barrier by measuring sodium fluorescein extravasation and blood vessel ultrastructure. In addition, tight junction markers, such as zona occludens-1, occludin and claudin-5, were quantified by western blot analysis. ZDF rats showed significantly increased sodium fluorescein leakage in the hippocampus. Tight junction markers, such as occludin and claudin-5, were significantly decreased in the hippocampi of ZDF rats compared to those of ZLC rats. In addition, ZDF rats showed ultrastructural changes with phagocytic findings in the blood vessels. These results suggest that chronic untreated diabetes impairs the permeability of the hippocampal blood-brain barrier by down-regulating occludin and claudin-5, indicating that chronic untreated diabetes may cause hippocampus-dependent dysfunction.

  18. Chronic type 2 diabetes reduces the integrity of the blood-brain barrier by reducing tight junction proteins in the hippocampus

    PubMed Central

    YOO, Dae Young; YIM, Hee Sun; JUNG, Hyo Young; NAM, Sung Min; KIM, Jong Whi; CHOI, Jung Hoon; SEONG, Je Kyung; YOON, Yeo Sung; KIM, Dae Won; HWANG, In Koo

    2016-01-01

    In the present study, we investigated the effects of type 2 diabetes-induced hyperglycemia on the integrity of the blood–brain barrier and tight junction markers in the rat hippocampus. Forty-week-old diabetic (Zucker diabetic fatty, ZDF) rats and littermate control (Zucker lean control, ZLC) rats were used in this study. We evaluated the integrity of the blood–brain barrier by measuring sodium fluorescein extravasation and blood vessel ultrastructure. In addition, tight junction markers, such as zona occludens-1, occludin and claudin-5, were quantified by western blot analysis. ZDF rats showed significantly increased sodium fluorescein leakage in the hippocampus. Tight junction markers, such as occludin and claudin-5, were significantly decreased in the hippocampi of ZDF rats compared to those of ZLC rats. In addition, ZDF rats showed ultrastructural changes with phagocytic findings in the blood vessels. These results suggest that chronic untreated diabetes impairs the permeability of the hippocampal blood–brain barrier by down-regulating occludin and claudin-5, indicating that chronic untreated diabetes may cause hippocampus-dependent dysfunction. PMID:26876499

  19. The expression patterns of tight junction protein claudin-1, -3, and -4 in human gastric neoplasms and adjacent non-neoplastic tissues

    PubMed Central

    Wang, Haiming; Yang, Xingwang

    2015-01-01

    Recently, there is growing evidence that tight junction proteins are often abnormally regulated in human tumors. The function of tight junction proteins in the maintenance of normal epithelial physiology has been well discussed, but their role in the tumorigenesis of gastric cancer is less well defined. To explore the expression distinction of the tight junction proteins claudin-1, -3, and -4 expression in the gastric cancer, the expression of claudin-1, -3, and -4 in 92 gastric cancer tissues and the non-neoplastic tissues adjacent to the tumors were examined by immunohistochemistry. Compared with adjacent non-neoplastic tissues, the expression of claudin-1 was down regulated. However, the expression of claudin-3 and claudin-4 were up-regulated in gastric cancer tissue. In addition, the expression of claudin-3 is correlated with claudin-4 expression in gastric cancer. Our present study reveals that claudin-1, -3, and -4 protein expression altered between human gastric cancers and adjacent non-neoplastic tissues. PMID:25755790

  20. TNF-α modulation of intestinal epithelial tight junction barrier is regulated by ERK1/2 activation of Elk-1.

    PubMed

    Al-Sadi, Rana; Guo, Shuhong; Ye, Dongmei; Ma, Thomas Y

    2013-12-01

    Tumor necrosis factor (TNF-α) is a proinflammatory cytokine that plays a critical role in the pathogenesis of inflammatory bowel disease. TNF-α causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of MAP kinase pathways (ERK1/2 and p38 kinase) and the molecular processes involved. An in vitro intestinal epithelial model system consisting of Caco-2 monolayers and an in vivo mouse model system were used to delineate the cellular and molecular mechanisms involved in TNF-α effects on tight junction barrier. The TNF-α-induced increase in Caco-2 tight junction permeability was mediated by activation of the ERK1/2 signaling pathway, but not the p38 kinase pathway. Activation of the ERK1/2 pathway led to phosphorylation and activation of the ETS domain-containing transcription factor Elk-1. The activated Elk-1 translocated to the nucleus, where it bound to its binding motif on the myosin light chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. In addition, in vivo intestinal perfusion studies also indicated that the TNF-α-induced increase in mouse intestinal permeability requires ERK1/2-dependent activation of Elk-1. These studies provide novel insight into the cellular and molecular processes that regulate the TNF-α-induced increase in intestinal epithelial tight junction permeability.

  1. The MAPK/ERK-Signaling Pathway Regulates the Expression and Distribution of Tight Junction Proteins in the Mouse Proximal Epididymis1

    PubMed Central

    Kim, Bongki; Breton, Sylvie

    2015-01-01

    The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. The MAPK/ERK1/2 pathway is maintained active in the IS by testicular luminal factors and plays crucial roles in the maintenance and differentiation of the IS epithelium. Tight junctions (TJs) are constituents of the blood-epididymis barrier, which mediates the paracellular transport of ions, solutes, and water and controls epithelial cell differentiation, thereby contributing to the establishment of a unique luminal environment. We examine here the role of the MAPK/ERK1/2 pathway in the regulation of TJ proteins in the IS. Inhibition of mitogen activated protein kinase kinase (MAPKK or MEK1/2) with PD325901, followed by reduction of ERK1/2 phosphorylation (pERK), decreased zonula occludens (ZO)-2 expression and increased ZO-3 expression in TJs but had no effect on ZO-1 expression. In control mice, in addition to being located in TJs, claudin (Cldn)-1, Cldn-3, and Cldn-4 were detected in the basolateral membrane of epithelial cells, with enriched expression of Cldn-1 and Cldn-4 in basal cells. PD325901 reduced the expression of Cldn-1 and Cldn-4 at all locations without affecting Cldn-3. Occludin was undetectable in the IS of control mice, but PD325901 triggered its expression in TJs. No effect was observed for any of the proteins examined in the other epididymal regions. Our results indicate the participation of the MAPK/ERK1/2 pathway in the regulation of cell-cell events that control the formation and maintenance of the blood-epididymis barrier. PMID:26658708

  2. Sodium Butyrate Promotes Reassembly of Tight Junctions in Caco-2 Monolayers Involving Inhibition of MLCK/MLC2 Pathway and Phosphorylation of PKCβ2

    PubMed Central

    Miao, Wei; Wu, Xiujuan; Wang, Kang; Wang, Wenjing; Wang, Yumei; Li, Zhigang; Liu, Jingjing; Li, Li; Peng, Luying

    2016-01-01

    As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection. PMID:27735862

  3. Transforming Growth Factor-β Regulation of Epithelial Tight Junction Proteins Enhances Barrier Function and Blocks Enterohemorrhagic Escherichia coli O157:H7-Induced Increased Permeability

    PubMed Central

    Howe, Kathryn L.; Reardon, Colin; Wang, Arthur; Nazli, Aisha; McKay, Derek M.

    2005-01-01

    Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an enteric pathogen that causes potentially fatal symptoms after intimate adhesion, modulation of intestinal epithelial signal transduction, and alteration of epithelial function (eg, barrier disruption). Although the epithelial barrier is critical to gut homeostasis, only a few agents, such as transforming growth factor (TGF)-β, can enhance or protect epithelial barrier function. Our aims were to delineate the mechanism(s) behind TGF-β-induced barrier enhancement and to determine whether TGF-β could prevent EHEC-induced barrier disruption. Using monolayers of the human T84 colonic epithelial cell line, we found that TGF-β induced a significant increase in transepithelial electrical resistance (a measure of paracellular permeability) through activation of ERK MAPK and SMAD signaling pathways and up-regulation of the tight junction protein claudin-1. Additionally, TGF-β pretreatment of epithelia blocked the decrease in transepithelial electrical resistance and the increase in transepithelial passage of [3H]-mannitol caused by EHEC infection. EHEC infection was associated with reduced expression of zonula occludens-1, occludin, and claudin-2 (but not claudin-1 or claudin-4); TGF-β pretreatment prevented these changes. These studies provide insight into EHEC pathogenesis by illustrating the mechanisms underlying TGF-β-induced epithelial barrier enhancement and identifying TGF-β as an agent capable of blocking EHEC-induced increases in epithelial permeability via maintenance of claudin-2, occludin, and zonula occludens-1 levels. PMID:16314472

  4. Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network

    PubMed Central

    Springler, Alexandra; Hessenberger, Sabine; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways. PMID:27618100

  5. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

    PubMed

    Song, Han; Zheng, Gang; Shen, Xue-Feng; Liu, Xin-Qin; Luo, Wen-Jing; Chen, Jing-Yuan

    2014-04-01

    Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.

  6. Studies of silicon pn junction solar cells

    NASA Technical Reports Server (NTRS)

    Lindholm, F. A.; Neugroschel, A.

    1977-01-01

    Modifications of the basic Shockley equations that result from the random and nonrandom spatial variations of the chemical composition of a semiconductor were developed. These modifications underlie the existence of the extensive emitter recombination current that limits the voltage over the open circuit of solar cells. The measurement of parameters, series resistance and the base diffusion length is discussed. Two methods are presented for establishing the energy bandgap narrowing in the heavily-doped emitter region. Corrections that can be important in the application of one of these methods to small test cells are examined. Oxide-charge-induced high-low-junction emitter (OCI-HLE) test cells which exhibit considerably higher voltage over the open circuit than was previously seen in n-on-p solar cells are described.

  7. Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells.

    PubMed

    Wuchter, Patrick; Boda-Heggemann, Judit; Straub, Beate K; Grund, Christine; Kuhn, Caecilia; Krause, Ulf; Seckinger, Anja; Peitsch, Wiebke K; Spring, Herbert; Ho, Anthony D; Franke, Werner W

    2007-06-01

    Substrate-adherent cultured cells derived from human bone marrow or umbilical cord blood ("mesenchymal stem cells") are of special interest for regenerative medicine. We report that such cells, which can display considerable heterogeneity with respect to their cytoskeletal protein complement, are often interconnected by special tentacle-like cell processes contacting one or several other cells. These processus adhaerentes, studded with many (usually small) puncta adhaerentia and varying greatly in length (up to more than 400 microm long), either contact each other in the intercellular space ("ET touches") or insert in a tight-fitting manner into deep plasma membrane invaginations (recessus adhaerentes), thus forming a novel kind of long (up to 50 microm) continuous cuff-like junction (manubria adhaerentia). The cell processes contain an actin microfilament core that is stabilized with ezrin, alpha-actinin, and myosin and accompanied by microtubules, and their adhering junctions are characterized by a molecular complement comprising the transmembrane glycoproteins N-cadherin and cadherin-11, in combination with the cytoplasmic plaque proteins alpha- and beta-catenin, together with p120(ctn), plakoglobin, and afadin. The processes are also highly dynamic and rapidly foreshorten as cell colonies approach a denser state of cell packing. These structures are obviously able to establish cell-cell connections, even over long distances, and can form deep-rooted and tight cell-cell adhesions. The possible relationship to similar cell processes in the embryonic primary mesenchyme and their potential in cell sorting and tissue formation processes in the body are discussed.

  8. Tight function zonula occludens-3 regulates cyclin D1-dependent cell proliferation.

    PubMed

    Capaldo, Christopher T; Koch, Stefan; Kwon, Michael; Laur, Oskar; Parkos, Charles A; Nusrat, Asma

    2011-05-15

    Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.

  9. The EhCPADH112 Complex of Entamoeba histolytica Interacts with Tight Junction Proteins Occludin and Claudin-1 to Produce Epithelial Damage

    PubMed Central

    Betanzos, Abigail; Javier-Reyna, Rosario; García-Rivera, Guillermina; Bañuelos, Cecilia; González-Mariscal, Lorenza; Schnoor, Michael; Orozco, Esther

    2013-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction. PMID:23762290

  10. Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat.

    PubMed

    Yang, Yi; Estrada, Eduardo Y; Thompson, Jeffrey F; Liu, Wenlan; Rosenberg, Gary A

    2007-04-01

    Matrix metalloproteinases (MMPs) disrupt the blood-brain barrier (BBB) during reperfusion. Occludin and claudins are recently described tight junction proteins (TJPs) that form the BBB. We hypothesized that the opening of the BBB was because of the degradation of TJPs by the MMPs. Spontaneously hypertensive rats had a 90 mins middle cerebral artery occlusion with reperfusion for 2, 3, or 24 h. Matrix metalloproteinases were measured by immunohistochemistry and in situ and gel zymography. Real-time polymerase chain reaction (PCR) measured mRNAs of MMP-2 and -9, furin, membrane-type MMP (MT1-MMP), occludin, and claudin-5. There was opening of the BBB in the piriform cortex after 3 h of reperfusion, and an MMP inhibitor, BB-1101 (30 mg/kg), prevented the opening. At 3 h, in situ zymograms showed gelatinase activity. Zymography and PCR showed greater increases in MMP-2 than in MMP-9. There were increased mRNA and immunohistochemistry for MT1-MMP and furin, which activate MMP-2. Claudin-5 and occludin mRNA expression decreased at 2 h in both hemispheres with fragments of both proteins seen on Western blot by 3 h on the ischemic side; treatment with BB-1101 reversed the degradation of the TJPs. Immunohistochemistry at 3 h showed fragmented TJPs within the endothelial cell clefts. By 24 h, in situ zymography showed gelatinase activity and gel zymography showed elevated levels of MMP-9. Disrupted TJPs previously seen in endothelial cells appeared in the surrounding astrocytes. Our results provide direct evidence that MMPs open the BBB by degrading TJPs and that an MMP inhibitor prevents degradation of the TJPs by MMPs.

  11. Moderate Hypoxia Followed by Reoxygenation Results in Blood-Brain Barrier Breakdown via Oxidative Stress-Dependent Tight-Junction Protein Disruption

    PubMed Central

    Zehendner, Christoph M.; Librizzi, Laura; Hedrich, Jana; Bauer, Nina M.; Angamo, Eskedar A.; de Curtis, Marco; Luhmann, Heiko J.

    2013-01-01

    Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in

  12. 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.

  13. cAMP Promotes Cell Migration Through Cell Junctional Complex Dynamics and Actin Cytoskeleton Remodeling: Implications in Skin Wound Healing.

    PubMed

    Kim, Mi Ok; Ryu, Jung Min; Suh, Han Na; Park, Soo Hyun; Oh, Yeon-Mok; Lee, Sang Hun; Han, Ho Jae

    2015-11-01

    Stem cells have attracted great interest for their therapeutic capacity in tissue regeneration. Cyclic adenosine 3',5'-monophosphate (cAMP), existing in high concentration at wound sites, mediated various signaling pathways such as cytoskeleton dynamics, cell adhesion, and cell migration in stem cells, which suggest the critical roles of cAMP in the wound healing process through functional regulation of stem cells. However, the mechanisms behind the effect of cAMP on mouse embryonic stem cell (mESC) motility and its roles on skin wound healing remain to be fully elucidated. In the present study, 8-Bromo cAMP-treated mESCs showed significant wound closure and improved neovascularization. Moreover, 8-Bromo cAMP stimulated mESC migration into the wound bed. 8-Bromo cAMP also increased ESC motility in in vitro migration assay. 8-Bromo cAMP induced myosin light chain phosphorylation through Rac1 and Cdc42 signaling, which were involved in 8-Bromo cAMP-induced decrease in expression of junction proteins (connexin 43, E-cadherin, and occludin) at the plasma membrane. Subsequently, 8-Bromo cAMP induced the disruption of cell junctions (including gap junctions, adherens junctions, and tight junctions), which reduced the function of the gap junctions and cell adhesion. In addition, 8-Bromo cAMP-induced Rac1 and Cdc42 activation increased Arp3, TOCA, PAK, and N-WASP expression, but decreased cofilin phosphorylation level, which elicited actin cytoskeleton remodeling. In contrast to the control, 8-Bromo cAMP evoked a substantial migration of cells into the denuded area, which was blocked by the small interfering RNAs of the signaling pathway-related molecules or by inhibitors. In conclusion, cAMP enhanced the migration of mESCs through effective coordination of junctional disruption and actin cytoskeleton remodeling, which increased the wound healing capacity of ESCs.

  14. Acanthopanax senticosus polysaccharides-induced intestinal tight junction injury alleviation via inhibition of NF-κB/MLCK pathway in a mouse endotoxemia model

    PubMed Central

    Han, Jie; Li, Ji-Hong; Bai, Guang; Shen, Guo-Shun; Chen, Jing; Liu, Jia-Nan; Wang, Shuo; Liu, Xian-Jun

    2017-01-01

    AIM To examine the effects of Acanthopanax senticosus polysaccharides (ASPS) on intestinal tight junction (TJ) disruption and nuclear factor-kappa B (NF-κB)/myosin light chain kinase (MLCK) activation in endotoxemia. METHODS BALB/C mice (6-8-weeks-old) received continuous intragastric gavage of ASPS for 7 d before injection of lipopolysaccharide (LPS), or received ASPS once after LPS injection. Blood and intestinal mucosal samples were collected 6 h after LPS challenge. Clinical symptoms, histological injury, intestinal permeability, TJ ultrastructure, and TJ protein expression were determined. RESULTS Compared with mice in the LPS group, pretreatment with ASPS improved clinical and histological scores by 390.9% (P < 0.05) and 57.89% (P < 0.05), respectively, and gut permeability change in endotoxemic mice was shown by a 61.93% reduction in reduced leakage of fluorescein isothiocyanate-dextran 6 h after LPS injection (P < 0.05). ASPS pretreatment also prevented LPS-induced TJ ultrastructure breakdown supported by increased electron dense materials between adjoining cells, sustained redistribution and expression of occludin (0.597 ± 0.027 vs 0.103 ± 0.009, P < 0.05) and zonula occludens-1 (0.507 ± 0.032 vs 0.125 ± 0.019, P < 0.05), and suppressed activation of the NF-κB/MLCK pathway indicated by reduced expression of NF-κB, phospho-inhibitor kappa B-alpha, MLCK and phospho-myosin light-chain-2 by 16.06% (P < 0.05), 54.31% (P < 0.05), 66.10% (P < 0.05) and 64.82% (P < 0.05), respectively. CONCLUSION ASPS pretreatment may be associated with inhibition of the NF-κB/MLCK pathway and concomitant amelioration of LPS-induced TJ dysfunction of intestinal epithelium in endotoxemia.

  15. ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro.

    PubMed

    Yang, Fuxing; Zhao, Kai; Zhang, Xiufeng; Zhang, Jun; Xu, Bainan

    2016-01-01

    Disruption of blood-brain barrier (BBB) follows brain trauma or central nervous system (CNS) stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R) by ATP induces the release of interleukin-1β (IL-1β), which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9). Degradation of tight junction proteins (TJPs) such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

  16. Polychlorinated biphenyls impair blood-brain barrier integrity via disruption of tight junction proteins in cerebrum, cerebellum and hippocampus of female Wistar rats: neuropotential role of quercetin.

    PubMed

    Selvakumar, K; Prabha, R Lakshmi; Saranya, K; Bavithra, S; Krishnamoorthy, G; Arunakaran, J

    2013-07-01

    Polychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties. Reactive oxygen species, which is produced from PCBs, alters blood-brain barrier (BBB) integrity, which is paralleled by cytoskeletal rearrangements and redistribution and disappearance of tight junction proteins (TJPs) like claudin-5 and occludin. Quercetin, a potent antioxidant present in onion and other vegetables, appears to protect brain cells against oxidative stress, a tissue-damaging process associated with Alzheimer's and other neurodegenerative disorders. The aim of this study is to analyze the role of quercetin on oxidative stress markers and transcription of transmembrane and cytoplasmic accessory TJPs on cerebrum, cerebellum and hippocampus of female rats exposed to PCBs. Rats were divided into the following four groups. Group I: received only vehicle (corn oil) intraperitoneally (i.p.); group II: received Aroclor 1254 at a dose of 2 mg/kg body weight (bwt)/day (i.p); group III: received Aroclor 1254 (i.p.) and simultaneously quercetin 50 mg/kg bwt/day through gavage and group IV: received quercetin alone gavage. From the experiment, the levels of hydrogen peroxide, lipid peroxidation and thiobarbituric acid reactive substances were observed to increase significantly in cerebrum, cerebellum and hippocampus as 50%, 25% and 20%, respectively, after exposure to PCB, and the messenger RNA expression of TJP in rats exposed to PCBs is decreased and is retrieved to the normal level simultaneously in quercetin-treated rats. Hence, quercetin can be used as a preventive medicine to PCBs exposure and prevents neurodegenerative disorders.

  17. Hirsutenone reduces deterioration of tight junction proteins through EGFR/Akt and ERK1/2 pathway both converging to HO-1 induction.

    PubMed

    Seo, Geom Seog; Jiang, Wen-Yi; Park, Pil-Hoon; Sohn, Dong Hwan; Cheon, Jae Hee; Lee, Sung Hee

    2014-07-15

    Oxidative stress-induced disruption of epithelial tight junctions (TJ) plays a critical role in the pathogenesis of intestinal disorders, including inflammatory bowel disease (IBD). The current study investigated the protective effect of hirsutenone against disruption of the intestinal barrier in vitro and in a mouse model of colitis. Caco-2 cells were stimulated with tert-butyl hydroperoxide (t-BH). Hirsutenone prevented the t-BH-induced increase in permeability by inhibiting the reduction in zonula occludens-1 (ZO-1) expression, and rapidly stimulated tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Hirsutenone-mediated protection against the loss of ZO-1 depends on the activation of both ERK1/2 and Akt signaling pathways. Interestingly, hirsutenone-mediated activation of Akt, but not ERK1/2, signaling was EGFR-dependent. Hirsutenone increased heme oxygenase-1 (HO-1) expression through both EGFR/Akt- and ERK1/2-dependent pathways, contributing to the protective effects against TJ dysfunction. Colitis was induced in mice by intrarectal administration of 2,4,6,-trinitrobenzene sulfonic acid (TNBS). Hirsutenone administration improved the clinical parameters and tissue histological appearance, increased HO-1 expression, attenuated reduction of ZO-1 and occludin mRNA, and promoted BrdU incorporation in the colonic epithelium of TNBS-treated mice. Taken together, our results demonstrate that hirsutenone reverse disordered intestinal permeability by activating EGFR/Akt and ERK1/2 pathways, which are involved in the regulation of HO-1 expression. These findings highlight the potential of hirsutenone for clinical applications in the treatment of IBD.

  18. Methamphetamine transiently increases the blood-brain barrier permeability in the hippocampus: role of tight junction proteins and matrix metalloproteinase-9.

    PubMed

    Martins, Tânia; Baptista, Sofia; Gonçalves, Joana; Leal, Ermelindo; Milhazes, Nuno; Borges, Fernanda; Ribeiro, Carlos F; Quintela, Oscar; Lendoiro, Elena; López-Rivadulla, Manuel; Ambrósio, António F; Silva, Ana P

    2011-09-09

    Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9.

  19. Activation of transient receptor potential vanilloid subtype 1 increases expression and permeability of tight junction in normal and hyposecretory submandibular gland.

    PubMed

    Cong, Xin; Zhang, Yan; Shi, Liang; Yang, Ning-Yan; Ding, Chong; Li, Jing; Ding, Qian-Wen; Su, Yun-Chao; Xiang, Ruo-Lan; Wu, Li-Ling; Yu, Guang-Yan

    2012-05-01

    Tight junction (TJ) is an important structure that regulates material transport through the paracellular pathway across the epithelium, but its significance in salivary physiology and pathogenesis of salivary dysfunctional diseases is not fully understood. We previously demonstrated that a functional transient receptor potential vanilloid subtype 1 (TRPV1) expresses in submandibular gland (SMG). However, association of TRPV1-induced saliva secretion with TJ remains unknown. Here we explored the effect of TRPV1 activation on expression and function of TJ of rabbit SMG in vitro and in vivo. RT-PCR and western blot analysis revealed that capsaicin upregulated expression of zonula occludin-1 (ZO-1), claudin (Cldn)-3, and -11, but not Cldn-1, -2, -4, -5, and -7 in cultured SMG cells. Capsaicin also increased the entering of 4 kDa FITC-dextran into the acinar lumen, induced redistribution of cytoskeleton F-actin under confocal microscope, and these effects were abolished by preincubation of capsazepine, a TRPV1 antagonist, indicating that activation of TRPV1 increases expression and permeability of TJ in SMG. Additionally, in a hyposecretory model induced by rabbit SMG transplantation, the expression of ZO-1, Cldn-3, and -11 was decreased, whereas other TJs remained unaltered. The structure of TJ was impaired and the width of apical TJs was reduced under transmission electron microscope, concomitant with diminished immunofluorescence of F-actin in peri-apicolateral region, indicating impaired TJ expression and decreased paracellular permeability in the transplanted SMG. Moreover, topical capsaicin cream increased secretion, decreased TJ structural injury, reversed TJ expression levels, and protected F-actin morphology from disarrangement in transplanted SMGs. These data provide the first evidence to demonstrate that TJ components, particularly ZO-1, Cldn-3, and -11 have important roles in secretion of SMG under both physiological and pathophysiological conditions. The

  20. ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro

    PubMed Central

    Zhao, Kai

    2016-01-01

    Disruption of blood-brain barrier (BBB) follows brain trauma or central nervous system (CNS) stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R) by ATP induces the release of interleukin-1β (IL-1β), which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9). Degradation of tight junction proteins (TJPs) such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9. PMID:27795859

  1. β-asarone and levodopa co-administration increase striatal dopamine level in 6-hydroxydopamine induced rats by modulating P-glycoprotein and tight junction proteins at the blood-brain barrier and promoting levodopa into the brain.

    PubMed

    Huang, Liping; Deng, Minzhen; He, Yuping; Lu, Shiyao; Ma, Ruanxin; Fang, Yongqi

    2016-06-01

    Levodopa (L-dopa) is widely considered as one of the most effective drug constituents in the treatment of Parkinson's disease (PD), but the blood-brain barrier (BBB) permeability of L-dopa is <5%, which causes low efficacy. Neuroprotective effects of β-asarone on 6-hydroxydopamine (6-OHDA)-induced PD rats were demonstrated by our previous studies. Co-administration of β-asarone and L-dopa has not been explored until being investigated on PD rats in this study. PD rats were divided into four groups: untreated, L-dopa-treated, β-asarone-treated and co-administered-treated groups. All of the treatments were administered to the rats twice per day for 30 days. The L-dopa, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), S100β and neuron-specific enolase (NSE) levels were subsequently determined. The P-glycoprotein (P-gp), zonula occludens-1 (ZO-1), claudin-5, occludin and actin expression was also assessed in cortex. Changes in BBB ultrastructure were observed using transmission electron microscopy. Our results showed that the co-administered treatment increased levels of L-dopa, DA, DOPAC and HVA in striatum, and S100β in plasma, but down-regulated NSE, P-gp, ZO-1, occludin, actin and claudin-5 in cortex. Crevices were observed between capillary endothelial cells at intercellular tight junction of the striatum in co-administered-treated group, while the endothelial cells in untreated group were tightly jointing each other. In addition, the correlations of L-dopa or DA and P-gp or tight junction proteins respectively were significantly negative in co-administered- and β-asarone-treated groups. These findings suggest that co-administered treatment may enhance the L-dopa BBB permeability and attenuate brain injury, which may be beneficial to PD treatment.

  2. 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

  3. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    SciTech Connect

    Zhang, Ying; Li, Jianguo

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

  4. Short communication: Glucagon-like peptide-2 and coccidiosis alter tight junction gene expression in the gastrointestinal tract of dairy calves.

    PubMed

    Walker, M P; Evock-Clover, C M; Elsasser, T H; Connor, E E

    2015-05-01

    Tight junction (TJ) proteins are integral factors involved in gut barrier function, and therapy with glucagon-like peptide-2 (GLP-2) enhances gut integrity. Our aim was to assess effects of GLP-2 treatment on mRNA expression of 8 TJ complex proteins in the intestine of dairy calves not infected or infected with Eimeria bovis at 11±3d of age. Mucosal epithelium from jejunum, ileum, and cecum was collected at slaughter from Holstein bull calves assigned to 4 groups: noninfected, buffer-treated (n=5); noninfected, GLP-2 treated (n=4); E. bovis-infected, buffer-treated (n=5); and E. bovis-infected, GLP-2-treated (n=4). Infected calves were orally dosed with 100,000 to 200,000 sporulated E. bovis oocysts on d 0; GLP-2-treated calves received 50 µg of GLP-2/kg of body weight subcutaneously twice daily for 10d beginning on d 18; and buffer-treated calves received an equal injection volume of 0.01 M Na bicarbonate buffer. All calves were killed on d 28. The mRNA expression of coxsackie and adenovirus receptor (CXADR), claudins 1, 2, and 4 (CLDN1, CLDN2, and CLDN4), F11 receptor (F11R), junction adhesion molecule 2 (JAM2), occludin (OCLN), and tight junction protein ZO-1 (TJP1) was determined by real-time quantitative PCR. In jejunum and ileum, an interaction of E. bovis infection and GLP-2 treatment on gene expression was noted. In jejunum of noninfected calves, GLP-2 increased CXADR, CLDN2, OCLN, and TJP1 mRNA expression but had no effect on mRNA expression in infected calves. Treatment with GLP-2 also increased tight junction protein ZO-1 protein expression in jejunum of noninfected calves as determined by immunohistochemistry. In ileum, E. bovis decreased expression of JAM2, OCLN, and TJP1 in buffer-treated calves, and GLP-2 increased TJP1 expression in infected calves. In cecum, E. bovis infection reduced expression of CXADR, CLDN4, F11R, and OCLN, and GLP-2 therapy increased expression of CLDN4, F11R, OCLN, and TJP1. Results are consistent with studies in

  5. Fabrication and analysis of dot junction silicon solar cells

    NASA Technical Reports Server (NTRS)

    Crotty, G. T.; Daud, T.

    1985-01-01

    A design of solar cells with reduced junction area on the cell surface is investigated for reduction of saturation current and increase in open-circuit voltage. Equidiameter dot junctions distributed across the surface of the cell offer an efficient alternative, with variations in dot diameter and in the spacing between dots giving the required variations in the ratio of junctions area to total surface area. A simplified analysis for short-circuit current and other cell parameters, which enables cell design optimization, is presented. Efficiencies beyond 18 percent are obtainable in flat-plate terrestrial applications. Experimental solar-cell performance results, as functions of different area ratios, and bulk doping are presented. It is shown that saturation current reduction and open-circuit voltage increase is obtained by reduced junction area.

  6. Studies of silicon p-n junction solar cells

    NASA Technical Reports Server (NTRS)

    Neugroschel, A.; Lindholm, F. A.

    1979-01-01

    To provide theoretical support for investigating different ways to obtain high open-circuit voltages in p-n junction silicon solar cells, an analytical treatment of heavily doped transparent-emitter devices is presented that includes the effects of bandgap narrowing, Fermi-Dirac statistics, a doping concentration gradient, and a finite surface recombination velocity at the emitter surface. Topics covered include: (1) experimental determination of bandgap narrowing in the emitter of silicon p-n junction devices; (2) heavily doped transparent regions in junction solar cells, diodes, and transistors; (3) high-low-emitter solar cell; (4) determination of lifetimes and recombination currents in p-n junction solar cells; (5) MOS and oxide-charged-induced BSF solar cells; and (6) design of high efficiency solar cells for space and terrestrial applications.

  7. Improved High/Low Junction Silicon Solar Cell

    NASA Technical Reports Server (NTRS)

    Neugroschel, A.; Pao, S. C.; Lindholm, F. A.; Fossum, J. G.

    1986-01-01

    Method developed to raise value of open-circuit voltage in silicon solar cells by incorporating high/low junction in cell emitter. Power-conversion efficiency of low-resistivity silicon solar cell considerably less than maximum theoretical value mainly because open-circuit voltage is smaller than simple p/n junction theory predicts. With this method, air-mass-zero opencircuit voltage increased from 600 mV level to approximately 650 mV.

  8. Molecular beam epitaxy growth of germanium junctions for multi-junction solar cell applications

    NASA Astrophysics Data System (ADS)

    Masuda, T.; Faucher, J.; Lee, M. L.

    2016-11-01

    We report on the molecular beam epitaxy (MBE) growth and device characteristics of Ge solar cells. Integrating a Ge bottom cell beneath a lattice-matched triple junction stack grown by MBE could enable ultra-high efficiencies without metamorphic growth or wafer bonding. However, a diffused junction cannot be readily formed in Ge by MBE due to the low sticking coefficient of group-V molecules on Ge surfaces. We therefore realized Ge junctions by growth of homo-epitaxial n-Ge on p-Ge wafers within a standard III-V MBE system. We then fabricated Ge solar cells, finding growth temperature and post-growth annealing to be key factors for achieving high efficiency. Open-circuit voltage and fill factor values of ~0.175 V and ~0.59 without a window layer were obtained, both of which are comparable to diffused Ge junctions formed by metal-organic vapor phase epitaxy. We also demonstrate growth of high-quality, single-domain GaAs on the Ge junction, as needed for subsequent growth of III-V subcells, and that the surface passivation afforded by the GaAs layer slightly improves the Ge cell performance.

  9. Elongated nanostructures for radial junction solar cells

    NASA Astrophysics Data System (ADS)

    Kuang, Yinghuan; Di Vece, Marcel; Rath, Jatindra K.; van Dijk, Lourens; Schropp, Ruud E. I.

    2013-10-01

    In solar cell technology, the current trend is to thin down the active absorber layer. The main advantage of a thinner absorber is primarily the reduced consumption of material and energy during production. For thin film silicon (Si) technology, thinning down the absorber layer is of particular interest since both the device throughput of vacuum deposition systems and the stability of the devices are significantly enhanced. These features lead to lower cost per installed watt peak for solar cells, provided that the (stabilized) efficiency is the same as for thicker devices. However, merely thinning down inevitably leads to a reduced light absorption. Therefore, advanced light trapping schemes are crucial to increase the light path length. The use of elongated nanostructures is a promising method for advanced light trapping. The enhanced optical performance originates from orthogonalization of the light's travel path with respect to the direction of carrier collection due to the radial junction, an improved anti-reflection effect thanks to the three-dimensional geometric configuration and the multiple scattering between individual nanostructures. These advantages potentially allow for high efficiency at a significantly reduced quantity and even at a reduced material quality, of the semiconductor material. In this article, several types of elongated nanostructures with the high potential to improve the device performance are reviewed. First, we briefly introduce the conventional solar cells with emphasis on thin film technology, following the most commonly used fabrication techniques for creating nanostructures with a high aspect ratio. Subsequently, several representative applications of elongated nanostructures, such as Si nanowires in realistic photovoltaic (PV) devices, are reviewed. Finally, the scientific challenges and an outlook for nanostructured PV devices are presented.

  10. Elongated nanostructures for radial junction solar cells.

    PubMed

    Kuang, Yinghuan; Vece, Marcel Di; Rath, Jatindra K; Dijk, Lourens van; Schropp, Ruud E I

    2013-10-01

    In solar cell technology, the current trend is to thin down the active absorber layer. The main advantage of a thinner absorber is primarily the reduced consumption of material and energy during production. For thin film silicon (Si) technology, thinning down the absorber layer is of particular interest since both the device throughput of vacuum deposition systems and the stability of the devices are significantly enhanced. These features lead to lower cost per installed watt peak for solar cells, provided that the (stabilized) efficiency is the same as for thicker devices. However, merely thinning down inevitably leads to a reduced light absorption. Therefore, advanced light trapping schemes are crucial to increase the light path length. The use of elongated nanostructures is a promising method for advanced light trapping. The enhanced optical performance originates from orthogonalization of the light's travel path with respect to the direction of carrier collection due to the radial junction, an improved anti-reflection effect thanks to the three-dimensional geometric configuration and the multiple scattering between individual nanostructures. These advantages potentially allow for high efficiency at a significantly reduced quantity and even at a reduced material quality, of the semiconductor material. In this article, several types of elongated nanostructures with the high potential to improve the device performance are reviewed. First, we briefly introduce the conventional solar cells with emphasis on thin film technology, following the most commonly used fabrication techniques for creating nanostructures with a high aspect ratio. Subsequently, several representative applications of elongated nanostructures, such as Si nanowires in realistic photovoltaic (PV) devices, are reviewed. Finally, the scientific challenges and an outlook for nanostructured PV devices are presented.

  11. Early weaning increases intestinal permeability, alters expression of cytokine and tight junction proteins, and activates mitogen-activated protein kinases in pigs.

    PubMed

    Hu, C H; Xiao, K; Luan, Z S; Song, J

    2013-03-01

    Although weaning stress has been reported to impair intestinal barrier function, the mechanisms have not yet been elucidated. In the present study, the intestinal morphology and permeability and mRNA expressions of tight junction proteins and cytokines in the intestine of piglets during the 2 wk after weaning were assessed. The phosphorylated (activated) ratios of p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular regulated kinases (ERK1/2) were determined to investigate whether mitogen-activated protein kinase (MAPK) signaling pathways are involved in the early weaning process. A shorter villus and deeper crypt were observed on d 3 and 7 postweaning. Although damaged intestinal morphology recovered to preweaning values on d 14 postweaning, the intestinal mucosal barrier, which was reflected by transepithelial electrical resistance (TER) and paracellular flux of dextran (4 kDa) in the Ussing chamber and tight junction protein expression, was not recovered. Compared with the preweaning stage (d 0), jejunal TER and mRNA expressions of occludin and claudin-1 on d 3, 7, and 14 postweaning and Zonula occludens-1 (ZO-1) mRNA on d 3 and 7 postweaning were reduced, and paracellular flux of dextran on d 3, 7, and 14 postweaning was increased. An increase (P < 0.05) of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA on d 3 and d 7 postweaning and an increase (P < 0.05) of interferon-γ (IFN-γ) mRNA on d 3 postweaning were observed compared with d 0. No significant increase of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) mRNA after weaning was observed. The phosphorylated (activated) ratios of JNK and p38 on d 3 and 7 postweaning and the phosphorylated ratio of ERK1/2 on d 3 postweaning were increased (P < 0.05) compared with d 0. The results indicated that early weaning induced sustained impairment in the intestinal barrier, decreased mRNA expression of tight junction proteins, and upregulated the expression of proinflammatory

  12. Reciprocal influence of connexins and apical junction proteins on their expressions and functions

    PubMed Central

    Derangeon, Mickaël; Spray, David C.; Bourmeyster, Nicolas; Sarrouilhe, Denis; Hervé, Jean-Claude

    2009-01-01

    Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another. PMID:19046940

  13. Expression of tight junction proteins and transporters for xenobiotic metabolism at the blood-CSF barrier during development in the nonhuman primate (P. hamadryas).

    PubMed

    Ek, C Joakim; D'Angelo, Barbara; Lehner, Christine; Nathanielsz, Peter; Li, Cun; Mallard, Carina

    2015-08-15

    The choroid plexus (CP) is rich in barrier mechanisms including transporters and enzymes which can influence drug disposition between blood and brain. We have limited knowledge of their state in fetus. We have studied barrier mechanisms along with metabolism and transporters influencing xenobiotics, using RNAseq and protein analysis, in the CP during the second-half of gestation in a nonhuman primate (Papio hamadryas). There were no differences in the expression of the tight-junctions at the CP suggesting a well-formed fetal blood-CSF barrier during this period of gestation. Further, the fetal CP express many enzymes for phase I-III metabolisms as well as transporters suggesting that it can greatly influence drug disposition and has a significant machinery to deactivate reactive molecules with only minor gestational changes. In summary, the study suggests that from, at least, midgestation, the CP in the nonhuman primate is restrictive and express most known genes associated with barrier function and transport.

  14. Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7

    SciTech Connect

    Mohandas, T.K.; Chen, X.N.; Korenberg, J.R.

    1995-12-10

    The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi X SPRET/Ei) F1 females X SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region. 13 refs., 2 figs.

  15. Histidine Prevents Cu-Induced Oxidative Stress and the Associated Decreases in mRNA from Encoding Tight Junction Proteins in the Intestine of Grass Carp (Ctenopharyngodon idella)

    PubMed Central

    Feng, Lin; Jiang, Jun; Kuang, Sheng-Yao; Wu, Pei; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2016-01-01

    Copper (Cu) is a common heavy metal pollutant in aquatic environments that originates from natural as well as anthropogenic sources. The present study investigated whether Cu causes oxidative damage and induces changes in the expression of genes that encode tight junction (TJ) proteins, cytokines and antioxidant-related genes in the intestine of the grass carp (Ctenopharyngodon idella). We demonstrated that Cu decreases the survival rate of fish and increases oxidative damage as measured by increases in malondialdehyde and protein carbonyl contents. Cu exposure significantly decreased the expression of genes that encode the tight junction proteins, namely, claudin (CLDN)-c, -3 and -15 as well as occludin and zonula occludens-1, in the intestine of fish. In addition, Cu exposure increases the mRNA levels of the pro-inflammatory cytokines, specifically, IL-8, TNF-α and its related signalling factor (nuclear factor kappa B, NF-κB), which was partly correlated to the decreased mRNA levels of NF-κB inhibitor protein (IκB). These changes were associated with Cu-induced oxidative stress detected by corresponding decreases in glutathione (GSH) content, as well as decreases in the copper, zinc-superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities and mRNA levels, which were associated with the down-regulated antioxidant signalling factor NF-E2-related factor-2 (Nrf2) mRNA levels, and the Kelch-like-ECH-associated protein1 (Keap1) mRNA levels in the intestine of fish. Histidine supplementation in diets (3.7 up to 12.2 g/kg) blocked Cu-induced changes. These results indicated that Cu-induced decreases in intestinal TJ proteins and cytokine mRNA levels might be partially mediated by oxidative stress and are prevented by histidine supplementation in fish diet. PMID:27280406

  16. Effect of dietary isoleucine on the immunity, antioxidant status, tight junctions and microflora in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian).

    PubMed

    Zhao, Juan; Feng, Lin; Liu, Yang; Jiang, Weidan; Wu, Pei; Jiang, Jun; Zhang, Yongan; Zhou, Xiaoqiu

    2014-12-01

    This study was conducted to investigate the effects of dietary isoleucine (Ile) on the immune response, antioxidant status, tight junctions, and microbial population in the intestine of juvenile Jian carp (Cyprinus carpio var. Jian). A total of 1200 juvenile Jian carp with average initial weight 6.9 ± 0.03 g were fed semi-purified isonitrogenous diets containing 4.2 (unsupplemented control group), 7.0, 9.5, 11.9, 13.9 and 16.9 g Ile kg(-1) diet for 60 days. Results indicated that Ile supplementation decreased malondialdehyde (MDA) and protein carbonyl content, and the amounts of Escherichia coli and Aeromonas in the intestine (P < 0.05), and increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), glutathione content and the amounts of Lactobacillus and Bacillus in the intestine (P < 0.05). Furthermore, real time polymerase chain reaction revealed that relative mRNA expression of copper/zinc superoxide dismutase (Cu-ZnSOD), manganese superoxide dismutase (MnSOD), CAT, NF-E2-related factor 2 (Nrf2), p38 mitogen-activated protein kinases (p38MAPK) in the intestine were increased with increasing of dietary Ile up to a certain point (P < 0.05). Conversely, the relative mRNA expression of occludin, claudin-3, claudin-7, TNF-α, IL-10, Kelch-like-ECH- associated protein 1 (Keap1), extracellular signal-regulated kinase 1 (ERK1) in the intestine showed a downward trend (P < 0.05). In conclusion, dietary Ile improves intestinal immune function, antioxidant capacity and microbial population, and regulates gene expression of antioxidant enzyme, tight junctions, Nrf2, Keap1, p38 and ERK1 in the intestine of Jian carp.

  17. Histidine Prevents Cu-Induced Oxidative Stress and the Associated Decreases in mRNA from Encoding Tight Junction Proteins in the Intestine of Grass Carp (Ctenopharyngodon idella).

    PubMed

    Jiang, Wei-Dan; Qu, Biao; Feng, Lin; Jiang, Jun; Kuang, Sheng-Yao; Wu, Pei; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Liu, Yang

    2016-01-01

    Copper (Cu) is a common heavy metal pollutant in aquatic environments that originates from natural as well as anthropogenic sources. The present study investigated whether Cu causes oxidative damage and induces changes in the expression of genes that encode tight junction (TJ) proteins, cytokines and antioxidant-related genes in the intestine of the grass carp (Ctenopharyngodon idella). We demonstrated that Cu decreases the survival rate of fish and increases oxidative damage as measured by increases in malondialdehyde and protein carbonyl contents. Cu exposure significantly decreased the expression of genes that encode the tight junction proteins, namely, claudin (CLDN)-c, -3 and -15 as well as occludin and zonula occludens-1, in the intestine of fish. In addition, Cu exposure increases the mRNA levels of the pro-inflammatory cytokines, specifically, IL-8, TNF-α and its related signalling factor (nuclear factor kappa B, NF-κB), which was partly correlated to the decreased mRNA levels of NF-κB inhibitor protein (IκB). These changes were associated with Cu-induced oxidative stress detected by corresponding decreases in glutathione (GSH) content, as well as decreases in the copper, zinc-superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities and mRNA levels, which were associated with the down-regulated antioxidant signalling factor NF-E2-related factor-2 (Nrf2) mRNA levels, and the Kelch-like-ECH-associated protein1 (Keap1) mRNA levels in the intestine of fish. Histidine supplementation in diets (3.7 up to 12.2 g/kg) blocked Cu-induced changes. These results indicated that Cu-induced decreases in intestinal TJ proteins and cytokine mRNA levels might be partially mediated by oxidative stress and are prevented by histidine supplementation in fish diet.

  18. Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

    PubMed Central

    Guo, Shuhong; Nighot, Meghali; Al-Sadi, Rana; Alhmoud, Tarik; Nighot, Prashant; Ma, Thomas Y.

    2015-01-01

    Gut-derived bacterial lipopolysaccharides (LPS) play an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). The defective intestinal tight junction (TJ) barrier has been shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, cause an increase in intestinal tight junction permeability (TJP) via a TLR-4 dependent process; however the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal TJ barrier using an in-vitro and in-vivo model system. LPS caused a TLR-4 dependent activation of membrane-associated adaptor protein FAK in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and –independent pathways. SiRNA silencing of MyD88 prevented LPS-induced increase in TJP. LPS caused a MyD88-dependent activation of IRAK4. TLR-4, FAK and MyD88 were co-localized. SiRNA silencing of TLR-4 inhibited TLR-4 associated FAK activation; and FAK knockdown prevented MyD88 activation. In-vivo studies also confirmed that LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented LPS-induced increase in intestinal permeability. Additionally, high dose LPS-induced intestinal inflammation was also dependent on TLR-4/FAK/MyD88 signal-transduction axis. Our data show for the first time that LPS-induced increase in intestinal TJP and intestinal inflammation was regulated by TLR-4 dependent activation of FAK-MyD88-IRAK4 signaling pathway. PMID:26466961

  19. Expression of tight junction molecule "claudins" in the lower oviductal segments and their changes with egg-laying phase and gonadal steroid stimulation in hens.

    PubMed

    Ariyadi, Bambang; Isobe, Naoki; Yoshimura, Yukinori

    2013-01-15

    Tight junctions in the mucosal epithelium have essential roles as a mucosal barrier to prevent invasion of microbes into the hen oviduct tissue. The aim of this study was to determine the effects of the egg-laying phase and estradiol on the expression of tight junction molecule "claudins" in the lower oviductal segments in hens. White Leghorn laying and molting hens were used. Molting hens were given either sesame oil (vehicle) or estradiol benzoate (N = 5 per group) via injection. The lower segments of oviduct (isthmus, uterus, and vagina) of these birds were collected. Gene expression of claudin-1, -3, -5, lipopolysaccharide-induced TNFα factor (LITAF), and IFN(γ) was analyzed by quantitative reverse transcription polymerase chain reaction, and localization of claudin-1 was examined by immunohistochemistry. Permeability in the mucosal epithelium was assessed by intrauterine injection of fluorescein isothiocyanate-dextran. Expression of claudin-1, -3, and -5 genes and density of claudin-1 protein in the lower oviductal segments were higher in laying hens than in molting hens (P < 0.01); their expression was upregulated by estradiol (P < 0.01). Expression of LITAF and IFN(γ) genes was higher in molting hens than in laying hens. More fluorescein isothiocyanate-dextran infiltrated into the intercellular space of the uterus mucosal epithelium in molting hens than in laying hens and estradiol-treated molting hens. In conclusion, we inferred that barrier functions of the mucosal epithelium in the lower oviductal segments might be disrupted because of reduced claudin expression in molting hens, which might increase the susceptibility of mucosal tissue during the molting phase.

  20. Full potential of radial junction Si thin film solar cells with advanced junction materials and design

    NASA Astrophysics Data System (ADS)

    Qian, Shengyi; Misra, Soumyadeep; Lu, Jiawen; Yu, Zhongwei; Yu, Linwei; Xu, Jun; Wang, Junzhuan; Xu, Ling; Shi, Yi; Chen, Kunji; Roca i Cabarrocas, Pere

    2015-07-01

    Combining advanced materials and junction design in nanowire-based thin film solar cells requires a different thinking of the optimization strategy, which is critical to fulfill the potential of nano-structured photovoltaics. Based on a comprehensive knowledge of the junction materials involved in the multilayer stack, we demonstrate here, in both experimental and theoretical manners, the potential of hydrogenated amorphous Si (a-Si:H) thin film solar cells in a radial junction (RJ) configuration. Resting upon a solid experimental basis, we also assess a more advanced tandem RJ structure with radially stacking a-Si:H/nanocrystalline Si (nc-Si:H) PIN junctions, and show that a balanced photo-current generation with a short circuit current density of Jsc = 14.2 mA/cm2 can be achieved in a tandem RJ cell, while reducing the expensive nc-Si:H absorber thickness from 1-3 μ m (in planar tandem cells) to only 120 nm. These results provide a clearly charted route towards a high performance Si thin film photovoltaics.

  1. Memory cell operation based on small Josephson junctions arrays

    NASA Astrophysics Data System (ADS)

    Braiman, Y.; Nair, N.; Rezac, J.; Imam, N.

    2016-12-01

    In this paper we analyze a cryogenic memory cell circuit based on a small coupled array of Josephson junctions. All the basic memory operations (e.g., write, read, and reset) are implemented on the same circuit and different junctions in the array can in principle be utilized for these operations. The presented memory operation paradigm is fundamentally different from conventional single quantum flux operation logics (SFQ). As an example, we demonstrate memory operation driven by a SFQ pulse employing an inductively coupled array of three Josephson junctions. We have chosen realistic Josephson junction parameters based on state-of-the-art fabrication capabilities and have calculated access times and access energies for basic memory cell operations. We also implemented an optimization procedure based on the simulated annealing algorithm to calculate the optimized and typical values of access times and access energies.

  2. Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs

    PubMed Central

    Ahn, Changhwan; Shin, Da-Hye; Lee, Dongoh; Kang, Su-Myung; Seok, Ju-Hyung; Kang, Hee Young; Jeung, Eui-Bae

    2016-01-01

    Tight junctions are the outermost structures of intercellular junctions and are classified as transmembrane proteins. These factors form selective permeability barriers between cells, act as paracellular transporters and regulate structural and functional polarity of cells. Although tight junctions have been previously studied, comparison of the transcriptional-translational levels of these molecules in canine organs remains to be investigated. In the present study, organ-specific expression of the tight junction proteins, claudin, occludin, junction adhesion molecule A and zona occludens 1 was examined in the canine duodenum, lung, liver and kidney. Results of immunohistochemistry analysis demonstrated that the tight junctions were localized in intestinal villi and glands of the duodenum, bronchiolar epithelia and alveolar walls of the lung, endometrium and myometrium of the hepatocytes, and the distal tubules and glomeruli of the kidney. These results suggest that tight junctions are differently expressed in organs, and therefore may be involved in organ-specific functions to maintain physiological homeostasis. PMID:27600198

  3. Improved GaAs solar cells with very thin junctions

    NASA Technical Reports Server (NTRS)

    Hovel, H. J.; Woodall, J. M.

    1976-01-01

    Violet cells with 500-1000 A junction depths have been made in GaAs by narrow junction diffusion followed by anodization. The best AM0 efficiencies obtained by this technique have been 10.5% (14% at AM1). GaAlAs-GaAs structures with very thin GaAlAs layers are much more promising, and efficiencies of over 18% at AM0 have been measured (21.9% at AM1).

  4. Cell accumulation in the junctional region of denervated muscle

    PubMed Central

    1987-01-01

    If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components. PMID:3491825

  5. 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

  6. Tunnel junctions for InP-on-Si solar cells

    NASA Technical Reports Server (NTRS)

    Keavney, C.; Vernon, S.; Haven, V.

    1991-01-01

    Growing, by metalorganic chemical vapor deposition, a tunnel junction is described, which makes possible and ohmic back contact in an n-on-p InP solar cell on a silicon substrate. The junction between heavily doped layers of p-type InGaAs and n-type InP shows resistance low enough not to affect the performance of these cells. InP solar cells made on n-type Si substrates with this structure were measured with an efficiency of 9.9 percent. Controls using p-type GaAs substrates showed no significant difference in cell performance, indicating that the resistance associated with the tunnel junction is less than about 0.1 ohm/sq cm.

  7. Development and fabrication of a solar cell junction processing system

    NASA Technical Reports Server (NTRS)

    1984-01-01

    A processing system capable of producing solar cell junctions by ion implantation followed by pulsed electron beam annealing was developed and constructed. The machine was to be capable of processing 4-inch diameter single-crystal wafers at a rate of 10(7) wafers per year. A microcomputer-controlled pulsed electron beam annealer with a vacuum interlocked wafer transport system was designed, built and demonstrated to produce solar cell junctions on 4-inch wafers with an AMI efficiency of 12%. Experiments showed that a non-mass-analyzed (NMA) ion beam could implant 10 keV phosphorous dopant to form solar cell junctions which were equivalent to mass-analyzed implants. A NMA ion implanter, compatible with the pulsed electron beam annealer and wafer transport system was designed in detail but was not built because of program termination.

  8. Non PN junction solar cells using carrier selective contacts

    NASA Astrophysics Data System (ADS)

    Bowden, Stuart; Ghosh, Kunal; Honsberg, Christiana

    2013-03-01

    A novel device concept utilizing the approach of selectively extracting carriers at the respective contacts is outlined in the work. The dominant silicon solar cell technology is based on a diffused, top-contacted p-n junction on a relatively thick silicon wafer for both commercial and laboratory solar cells. The VOC and hence the efficiency of a diffused p-n junction solar cell is limited by the emitter recombination current and a value of 720 mV is considered to be the upper limit. The value is more than 100 mV smaller than the thermodynamic limit of VOC as applicable for silicon based solar cells. Also, in diffused junction the use of thin wafers (< 50 um) are problematic because of the requirement of high temperature processing steps. But a number of roadmaps have identified solar cells manufactured on thinner silicon wafers to achieve lower cost and higher efficiency. The carrier selective contact device provides a novel alternative to diffused p-n junction solar cells by eliminating the need for complementary doping to form the emitter and hence it allows the solar cells to achieve a VOC of greater than 720 mV. Also, the complete device structure can be fabricated with low temperature thin film deposition or organic coating on silicon substrates and thus epitaxially grown silicon or kerfless silicon, in addition to standard silicon wafers can be utilized.

  9. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet.

    PubMed

    Elfers, Kristin; Marr, Isabell; Wilkens, Mirja R; Breves, Gerhard; Langeheine, Marion; Brehm, Ralph; Muscher-Banse, Alexandra S

    2016-01-01

    Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.

  10. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intest