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

PubMed

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

2015-02-01

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

MarvelD3 couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival

PubMed Central

Steed, Emily; Elbediwy, Ahmed; Vacca, Barbara; Dupasquier, Sébastien; Hemkemeyer, Sandra A.; Suddason, Tesha; Costa, Ana C.; Beaudry, Jean-Bernard; Zihni, Ceniz; Gallagher, Ewen; Pierreux, Christophe E.

2014-01-01

MarvelD3 is a transmembrane component of tight junctions, but there is little evidence for a direct involvement in the junctional permeability barrier. Tight junctions also regulate signaling mechanisms that guide cell proliferation; however, the transmembrane components that link the junction to such signaling pathways are not well understood. In this paper, we show that MarvelD3 is a dynamic junctional regulator of the MEKK1–c-Jun NH2-terminal kinase (JNK) pathway. Loss of MarvelD3 expression in differentiating Caco-2 cells resulted in increased cell migration and proliferation, whereas reexpression in a metastatic tumor cell line inhibited migration, proliferation, and in vivo tumor formation. Expression levels of MarvelD3 inversely correlated with JNK activity, as MarvelD3 recruited MEKK1 to junctions, leading to down-regulation of JNK phosphorylation and inhibition of JNK-regulated transcriptional mechanisms. Interplay between MarvelD3 internalization and JNK activation tuned activation of MEKK1 during osmotic stress, leading to junction dissociation and cell death in MarvelD3-depleted cells. MarvelD3 thus couples tight junctions to the MEKK1–JNK pathway to regulate cell behavior and survival. PMID:24567356

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

PubMed Central

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

2007-01-01

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

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

PubMed

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

2007-11-20

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

Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis.

PubMed

Wang, Jiang; Li, Chang; Jiang, Yingjian; Zheng, Hongmei; Li, Dehui; Liang, Yibo; Deng, Wensheng; Zhang, Dianliang

2017-02-01

The aim of the study was to investigate the effects of ceramide-1-phosphate transfer protein (CPTP) on the intestinal epithelial tight junction proteins in patients with severe acute pancreatitis (SAP). Fifty patients with SAP were classified into two groups according to the presence of bacterial translocation (BT) in the blood. Thirty healthy individuals were included in the control group. The presence of BT was analyzed by polymerase chain reaction. The expression of tight junction proteins and CPTP was determined using immunohistochemistry and western blotting. Bacterial DNA was detected in the peripheral blood of 62.0% of the patients with SAP. The expression of CPTP and tight junction proteins in SAP patients was lower than that in healthy controls. Among the patients with SAP, those positive for BT(+) showed a lower level of CPTP and occluding (OC) and zonula occludens-1 (ZO-1) expression and a higher level of IVA cPLA2 expression than BT(-) patients. Moreover, the expression of CPTP was significantly associated with ZO-1 and showed a negative correlation with expression of IVA cPLA2 in SAP-BT(+) patients. CPTP affects the expression of tight junction proteins and may protects the intestinal epithelial barrier by downregulating the expression of IVA cPLA2. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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.

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

PubMed

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

2017-10-01

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

Dietary glucosylceramide enhances tight junction function in skin epidermis via induction of claudin-1.

PubMed

Kawada, Chinatsu; Hasegawa, Tatsuya; Watanabe, Mutsuto; Nomura, Yoshihiro

2013-01-01

Dietary glucosylceramide increased the expression of claudin-1 in UVB-irradiated mouse epidermis. Sphingosine and phytosphingosine, metabolites of glucosylceramide, increased trans-epithelial electrical resistance, and phytosphingosine increased claudin-1 mRNA expression in cultured keratinocytes. Our results indicate that the skin barrier improvement induced by dietary glucosylceramide might be due to enhancement of tight junction function, mediated by increased expression of claudin-1 induced by sphingoid metabolites.

Tricellulin, occludin and claudin-3 expression in salmon intestine and kidney during salinity adaptation.

PubMed

Tipsmark, C K; Madsen, S S

2012-08-01

Molecular regulation of tight junctions in osmoregulatory epithelia of euryhaline fishes must be extensive during ontogeny and acclimation to salinity changes. In this study, five tight junction proteins were examined in Atlantic salmon (Salmo salar): tight junction associated tricellulin, occludin and claudin-3 isoforms (a, b, c). A survey of tissue distribution in freshwater (FW) salmon showed that tricellulin expression was highest in the intestine. Occludin was detected in tissues with importance for epithelial transport and the order of expression was gill>intestine>kidney. The three claudin-3 isoforms were expressed at highest level in kidney tissue. Transfer of juvenile FW salmon to seawater (SW) elevated intestinal tricellulin and occludin mRNA, and these transcripts were also elevated at the time of best SW-tolerance during the course of smoltification. In the kidney, expression of tricellulin and claudin-3 isoforms was elevated after SW-transfer and tricellulin, occludin, claudin-3a and -3b increased in March before the peak smolt stage. In the gill, none of the examined tight junction proteins were impacted by SW-transfer. The data suggest that expression of tricellulin and occludin is dynamically involved in reorganization of intestinal epithelium and possibly changed paracellular permeability during SW-acclimation. The increased renal tricellulin and claudin-3 expression in SW suggests a role in remodeling of the kidney during SW-acclimation. Copyright © 2012 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2011-01-01

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

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.

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.

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

PubMed

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

2015-07-08

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

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

PubMed

Vetrano, Stefania; Danese, Silvio

2009-05-01

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

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

PubMed Central

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

2009-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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

IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

PubMed Central

Wise, Sarah K.; Laury, Adrienne M.; Katz, Elizabeth H.; Den Beste, Kyle A.; Parkos, Charles A.; Nusrat, Asma

2014-01-01

Introduction Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a “leaky” epithelial barrier phenotype. We hypothesize that Th2 cytokines IL-4 and IL-13 modulate epithelial junction proteins thereby contributing to the leaky epithelial barrier. Methods Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. Results IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n=6) and 68.6% (n=8) of baseline, respectively. Tight junction protein JAM-A expression decreased 42.2% with IL-4 exposure (n=9) and 37.5% with IL-13 exposure (n=9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n=9) and 32.9% with IL-13 exposure (n=9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or ZO-1 with IL-4 or IL-13 exposure. Conclusion Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. PMID:24510479

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

PubMed

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

2012-12-13

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

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

PubMed

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

2011-06-01

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

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

PubMed

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

2018-06-27

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

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

PubMed

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

1984-12-01

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

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

PubMed

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

2015-04-01

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

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

PubMed

Wise, Sarah K; Laury, Adrienne M; Katz, Elizabeth H; Den Beste, Kyle A; Parkos, Charles A; Nusrat, Asma

2014-05-01

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity. © 2014 ARS-AAOA, LLC.

EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

PubMed

Ding, Gui-Rong; Qiu, Lian-Bo; Wang, Xiao-Wu; Li, Kang-Chu; Zhou, Yong-Chun; Zhou, Yan; Zhang, Jie; Zhou, Jia-Xing; Li, Yu-Rong; Guo, Guo-Zhen

2010-07-15

The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Physiological roles of claudins in kidney tubule paracellular transport.

PubMed

Muto, Shigeaki

2017-01-01

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

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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2011-11-01

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

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

PubMed

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

2017-10-01

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

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2013-08-01

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

Tight junction physiology of pleural mesothelium

PubMed Central

Markov, Alexander G.; Amasheh, Salah

2014-01-01

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

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

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

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

2007-05-15

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

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

PubMed

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

2010-07-02

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

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

PubMed

Yang, Shu; Jin, Hong; Zhao, Zhigang

2018-04-23

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

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

PubMed Central

Peng, Shaomin; Adelman, Ron A.

2010-01-01

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

[Effects of electromagnetic pulse on blood-brain barrier permeability and tight junction proteins in rats].

PubMed

Qiu, Lian-bo; Ding, Gui-rong; Zhang, Ya-mei; Zhou, Yan; Wang, Xiao-wu; Li, Kang-chu; Xu, Sheng-long; Tan, Juan; Zhou, Jia-xing; Guo, Guo-zhen

2009-09-01

To study the effect of electromagnetic pulse (EMP) on the permeability of blood-brain barrier, tight junction (TJ)-associated protein expression and localization in rats. 66 male SD rats, weighing (200 approximately 250) g, were sham or whole-body exposed to EMP at 200 kV/m for 200 pulses. The repetition rate was 1 Hz. The permeability of the blood-brain barrier in rats was assessed by albumin immunohistochemistry. The expression of typical tight junction protein ZO-1 and occludin in both cerebral cortex homogenate and cerebral cortex microvessel homogenate was analyzed by the Western blotting and the distribution of ZO-1 and occludin was examined by immunofluorescence microscopy. In the sham exposure rats, no brain capillaries showed albumin leakage, at 0.5 h after 200 kV/m EMP exposure for 200 pulses; a few brain capillaries with extravasated serum albumin was found, with the time extended, the number of brain capillaries with extravasated serum albumin increased, and reached the peak at 3 h, then began to recover at 6 h. In addition, no change in the distribution of the occludin was found after EMP exposure. Total occludin expression had no significant change compared with the control. However, the expression level of ZO-1 significantly decreased at 1 h and 3 h after EMP exposure in both cerebral cortex homogenate and cerebral cortex microvessel homogenate. Furthermore, immunofluorescence studies also showed alterations in ZO-1 protein localization in cerebral cortex microvessel. The EMP exposure (200 kV/m, 200 pulses) could increase blood-brain barrier permeability in rat, and this change is associated with specific alterations in tight junction protein ZO-1.

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

PubMed

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

2016-10-01

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

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

PubMed Central

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

2014-01-01

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

Decreased expression of zonula occludens-1 and occludin in the bladder urothelium of patients with interstitial cystitis/painful bladder syndrome.

PubMed

Lee, Jane-Dar; Lee, Ming-Huei

2014-01-01

Unique barrier properties of the urothelial surface membrane permit urine storage without contents leak into the bloodstream. Previous reports suggested that the bladder urothelial barrier might be compromised in interstitial cystitis/painful bladder syndrome (IC/PBS). We examined the changes of tight junction proteins (zonula occludens-1 (ZO-1) and occludin) in IC/PBS patients. Bladder samples were derived from of 32 patients with IC/PBS and eight controls. We detected the tight junction proteins of ZO-1 and occludin expression by immunoblotting, immunohistochemical (IHC) staining and double immunofluorescent (IF) staining with confocal microscopy. Data were analyzed using the Mann-Whitney U-test. Expression of ZO-1 and occludin in the IC/PBS group was reduced compared to the control group by immunoblotting and IHC staining. Also, the thinning and denudation of urothelium were demonstrated in the IC/PBS group by histological study. IF staining showed the interruption of bladder urothelium in IC/PBS patients under confocal microscopy. Our data showed that decreased expression of tight junction proteins (ZO-1 and occludin) and interruption of bladder urothelium in IC/PBS patients. Treatment to repair the discontinuous urothelium may be useful to relieve some clinical symptoms of patients with IC/PBS. Copyright © 2012. Published by Elsevier B.V.

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

PubMed

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

2018-01-01

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

Tight junctions in cancer metastasis.

PubMed

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

2011-01-01

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

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

PubMed

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

2013-12-01

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

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

NASA Astrophysics Data System (ADS)

Gordon, Geoffrey; Lo, Chun-Min

2007-03-01

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

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

PubMed

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

2008-06-01

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

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

PubMed Central

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

2008-01-01

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

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

PubMed

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

2016-10-28

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

Hampl, R; Bičíková, M; Sosvorová, L

2015-01-01

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

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

PubMed Central

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

2011-01-01

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

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

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

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

PubMed

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

2015-12-01

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

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

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

Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret

2011-07-08

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

Isoflurane anesthesia results in reversible ultrastructure and occludin tight junction protein expression changes in hippocampal blood-brain barrier in aged rats.

PubMed

Cao, Yiyun; Ni, Cheng; Li, Zhengqian; Li, Lunxu; Liu, Yajie; Wang, Chunyi; Zhong, Yanfeng; Cui, Dehua; Guo, Xiangyang

2015-02-05

The underlying mechanism of isoflurane-induced cognitive dysfunction in older individuals is unknown. In this study, the effects of isoflurane exposure on the hippocampal blood-brain barrier (BBB) in aged rats were investigated because it was previously shown that BBB disruption involves in cognitive dysfunction. Twenty-month-old rats randomly received 1.5% isoflurane or vehicle gas as control. Hippocampal BBB ultrastructure was analyzed by transmission electron microscopy and expression of tight junction proteins was measured by western blot analysis. BBB permeability was detected with sodium fluorescein extravasation and further confirmed by immunoglobulin G immunohistochemistry. Spatial learning and memory were assessed by the Morris water maze test. Isoflurane anesthesia resulted in reversible time-dependent BBB ultrastructure morphological damage and significant decreases in expression of the tight junction proteins occludin, which contributed to sodium fluorescein and IgG leakage. Rats with isoflurane exposure also showed significant cognitive deficits in the Morris water maze test. This in vivo data indicate that occludin down-regulation may be one of the mediators of isoflurane-induced hippocampus BBB disruption, and may contribute to hippocampus-dependent cognitive impairment after isoflurane exposure in aged rats. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2018-06-01

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

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

PubMed Central

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

2012-01-01

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

Tight junctions and human diseases.

PubMed

Sawada, Norimasa; Murata, Masaki; Kikuchi, Keisuke; Osanai, Makoto; Tobioka, Hirotoshi; Kojima, Takashi; Chiba, Hideki

2003-09-01

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

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

PubMed

Ikemura, Kenji; Iwamoto, Takuya; Okuda, Masahiro

2014-08-01

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

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

PubMed Central

Cohen, L; Sekler, I; Hershfinkel, M

2014-01-01

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

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

PubMed

Cohen, L; Sekler, I; Hershfinkel, M

2014-06-26

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

Blood-Brain Barrier Permeability Is Exacerbated in Experimental Model of Hepatic Encephalopathy via MMP-9 Activation and Downregulation of Tight Junction Proteins.

PubMed

Dhanda, Saurabh; Sandhir, Rajat

2018-05-01

The present study was designed to investigate the mechanisms involved in blood-brain barrier (BBB) permeability in bile duct ligation (BDL) model of chronic hepatic encephalopathy (HE). Four weeks after BDL surgery, a significant increase was observed in serum bilirubin levels. Masson trichrome staining revealed severe hepatic fibrosis in the BDL rats. 99m Tc-mebrofenin retention was increased in the liver of BDL rats suggesting impaired hepatobiliary transport. An increase in permeability to sodium fluorescein, Evans blue, and fluorescein isothiocyanate (FITC)-dextran along with increase in water and electrolyte content was observed in brain regions of BDL rats suggesting disrupted BBB. Increased brain water content can be attributed to increase in aquaporin-4 mRNA and protein expression in BDL rats. Matrix metalloproteinase-9 (MMP-9) mRNA and protein expression was increased in brain regions of BDL rats. Additionally, mRNA and protein expression of tissue inhibitor of matrix metalloproteinases (TIMPs) was also increased in different regions of brain. A significant decrease in mRNA expression and protein levels of tight junction proteins, viz., occludin, claudin-5, and zona occluden-1 (ZO-1) was observed in different brain regions of BDL rats. VCAM-1 mRNA and protein expression was also found to be significantly upregulated in different brain regions of BDL animals. The findings from the study suggest that increased BBB permeability in HE involves activation of MMP-9 and loss of tight junction proteins.

Tight junctions and the modulation of barrier function in disease

PubMed Central

2008-01-01

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease. PMID:18415116

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

PubMed Central

Zuhl, Micah N.; Moseley, Pope L.

2015-01-01

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

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

PubMed

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

2016-03-15

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

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 junction disruption and barrier dysfunction.

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

PubMed

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

2016-02-01

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

Comparison of Tight Junction Protein-Related Gene mRNA Expression Levels between Male and Female Gastroesophageal Reflux Disease Patients.

PubMed

Kim, Jin Joo; Kim, Nayoung; Park, Ji Hyun; Kim, Young Sun; Lee, Sun Min; Lee, Dong Ho; Jung, Hyun Chae

2018-03-21

Male predominance has been observed in the erosive reflux disease (ERD), but reverse finding in nonerosive reflux disease (NERD). This suggests sex-specific medicine approach is needed but its mechanism is remained to be elucidated. We aimed to compare clinical characteristics and mRNA expression levels of tight junction-related proteins between male and female gastroesophageal reflux disease (GERD) patients. Sixteen healthy controls, 45 ERD, and 14 NERD patients received upper endoscopies and completed questionnaires. Quantitative real-time polymerase chain reactions (qPCR) of occludin (OCLN), zonal occludens (ZO) 1, claudin1 (CDLN1) and claudin4 (CDLN4), and neurokinin 1 receptor (NK1R) were performed in the distal esophageal mucosal specimen. These results were analyzed by sex. Female GERD patients were affected more by reflux symptoms than males. The impairment of overall QoL was more prominent in female patients with reflux symptoms than male patients (5.6±0.2 vs. 4.9±0.6, p=0.009). The levels of OCLN mRNA expression were significantly lower in the male ERD group. On the other hand, those of CLDN1, CLDN4, and NK1R except ZO-1 were significantly higher in the male ERD group. We demonstrated that female ERD/NERD patients were affected more by GERD and male ERD patients showed significant changes of tight junction protein mRNA expression levels.

Blood-brain Barrier Disruption Leads to Postoperative Cognitive Dysfunction.

PubMed

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

2017-01-01

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

Additive Effects of Rebamipide Plus Proton Pump Inhibitors on the Expression of Tight Junction Proteins in a Rat Model of Gastro-Esophageal Reflux Disease.

PubMed

Gweon, Tae-Geun; Park, Jong-Hyung; Kim, Byung-Wook; Choi, Yang Kyu; Kim, Joon Sung; Park, Sung Min; Kim, Chang Whan; Kim, Hyung-Gil; Chung, Jun-Won

2018-01-15

The aim of this study was to investigate the effects of rebamipide on tight junction proteins in the esophageal mucosa in a rat model of gastroesophageal reflux disease (GERD). GERD was created in rats by tying the proximal stomach. The rats were divided into a control group, a proton pump inhibitor (PPI) group, and a PPI plus rebamipide (PPI+R) group. Pantoprazole (5 mg/kg) was administered intraperitoneally to the PPI and PPI+R groups. An additional dose of rebamipide (100 mg/kg) was administered orally to the PPI+R group. Mucosal erosions, epithelial thickness, and leukocyte infiltration into the esophageal mucosa were measured in isolated esophagi 14 days after the procedure. A Western blot analysis was conducted to measure the expression of claudin-1, -3, and -4. The mean surface area of mucosal erosions, epithelial thickness, and leukocyte infiltration were lower in the PPI group and the PPI+R group than in the control group. Western blot analysis revealed that the expression of claudin-3 and -4 was significantly higher in the PPI+R group than in the control group. Rebamipide may exert an additive effect in combination with PPI to modify the tight junction proteins of the esophageal mucosa in a rat model of GERD. This treatment might be associated with the relief of GERD symptoms.

Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

PubMed

Kim, Yeojung; Kessler, Sean P; Obery, Dana R; Homer, Craig R; McDonald, Christine; de la Motte, Carol A

2017-10-01

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis

PubMed Central

Bardet, Claire; Ribes, Sandy; Wu, Yong; Diallo, Mamadou Tidiane; Salmon, Benjamin; Breiderhoff, Tilman; Houillier, Pascal; Müller, Dominik; Chaussain, Catherine

2017-01-01

Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis. PMID:28596736

Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis.

PubMed

Bardet, Claire; Ribes, Sandy; Wu, Yong; Diallo, Mamadou Tidiane; Salmon, Benjamin; Breiderhoff, Tilman; Houillier, Pascal; Müller, Dominik; Chaussain, Catherine

2017-01-01

Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis.

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

PubMed

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

2012-11-01

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

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

Transmembrane proteins of tight junctions.

PubMed

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

2008-03-01

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

Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue.

PubMed

Hatte, Guillaume; Prigent, Claude; Tassan, Jean-Pierre

2018-02-05

Epithelia are layers of polarised cells tightly bound to each other by adhesive contacts. Epithelia act as barriers between an organism and its external environment. Understanding how epithelia maintain their essential integrity while remaining sufficiently plastic to allow events such as cytokinesis to take place is a key biological problem. In vertebrates, the remodelling and reinforcement of adherens junctions maintains epithelial integrity during cytokinesis. The involvement of tight junctions in cell division, however, has remained unexplored. Here, we examine the role of tight junctions during cytokinesis in the epithelium of the Xenopus laevis embryo. Depletion of the tight junction-associated proteins ZO-1 and GEF-H1 leads to altered cytokinesis duration and contractile ring geometry. Using a tension biosensor, we show that cytokinesis defects originate from misregulation of tensile forces applied to adherens junctions. Our results reveal that tight junctions regulate mechanical tension applied to adherens junctions, which in turn impacts cytokinesis.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

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

Effects of metformin treatment on glioma-induced brain edema

PubMed Central

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

2016-01-01

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

Effects of metformin treatment on glioma-induced brain edema.

PubMed

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

2016-01-01

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

Glutamine enhances tight junction protein expression and modulates corticotropin-releasing factor signaling in the jejunum of weanling piglets.

PubMed

Wang, Hao; Zhang, Chen; Wu, Guoyao; Sun, Yuli; Wang, Bin; He, Beibei; Dai, Zhaolai; Wu, Zhenlong

2015-01-01

Dysfunction of tight junction integrity is associated with decreased nutrient absorption and numerous gastrointestinal diseases in humans and piglets. Although l-glutamine has been reported to enhance intestinal-mucosal mass and barrier function under stressful conditions, in vivo data to support a functional role for l-glutamine on intestinal tight junction protein (TJP) expression in weanling mammals are limited. This study tested the hypothesis that glutamine regulates expression of TJPs and stress-related corticotropin-releasing factor (CRF) signaling in the jejunum of weanling piglets. Piglets were reared by sows or weaned at 21 d of age to a corn and soybean meal-based diet that was or was not supplemented with 1% l-glutamine for 7 d. Growth performance, intestinal permeability, TJP abundance, and CRF expression were examined. Weaning caused increases (P < 0.05) in intestinal permeability by 40% and in CRF concentrations by 4.7 times in association with villus atrophy (P < 0.05). Western blot analysis showed reductions (P < 0.05) in jejunal expression of occludin, claudin-1, zonula occludens (ZO) 2, and ZO-3, but no changes in the abundance of claudin-3, claudin-4, or ZO-1 in weanling piglets compared with age-matched suckling controls. Glutamine supplementation improved (P < 0.05) intestinal permeability and villus height, while reducing (P < 0.05) jejunal mRNA and protein levels for CRF and attenuating (P < 0.05) weanling-induced decreases in occludin, claudin-1, ZO-2, and ZO-3 protein abundances. Collectively, our results support an important role for l-glutamine in regulating expression of TJPs and CRF in the jejunum of weanling piglets. © 2015 American Society for Nutrition.

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

PubMed Central

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

2010-01-01

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

Holding Tight: Cell Junctions and Cancer Spread.

PubMed

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

2012-01-01

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

On the self-association potential of transmembrane tight junction proteins.

PubMed

Blasig, I E; Winkler, L; Lassowski, B; Mueller, S L; Zuleger, N; Krause, E; Krause, G; Gast, K; Kolbe, M; Piontek, J

2006-02-01

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

Electronic transport across a junction between armchair graphene nanotube and zigzag nanoribbon. Transmission in an armchair nanotube without a zigzag half-line of dimers

NASA Astrophysics Data System (ADS)

Sharma, Basant Lal

2018-05-01

Based on the well known nearest-neighbor tight-binding approximation for graphene, an exact expression for the electronic conductance across a zigzag nanoribbon/armchair nanotube junction is presented for non-interacting electrons. The junction results from the removal of a half-row of zigzag dimers in armchair nanotube, or equivalently by partial rolling of zigzag nanoribbon and insertion of a half-row of zigzag dimers in between. From the former point of view, a discrete form of Dirichlet condition is imposed on a zigzag half-line of dimers assuming the vanishing of wave function outside the physical structure. A closed form expression is provided for the reflection and transmission moduli for the outgoing wave modes for each given electronic wave mode incident from either side of the junction. It is demonstrated that such a contact junction between the nanotube and nanoribbon exhibits negligible backscattering, and the transmission has been found to be nearly ballistic. In contrast to the previously reported studies for partially unzipped carbon nanotubes (CNTs), using the same tight binding model, it is found that due to the "defect" there is certain amount of mixing between the electronic wave modes with even and odd reflection symmetries. But the junction remains a perfect valley filter for CNTs at certain energy ranges. Applications aside from the electronic case, include wave propagation in quasi-one-dimensional honeycomb structures of graphene-like constitution. The paper includes several numerical calculations, analytical derivations, and graphical results, which complement the provision of succinct closed form expressions.

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

PubMed

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

2009-08-01

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

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

DOE PAGES

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

2016-08-09

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

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

PubMed Central

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

2009-01-01

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

Gap and tight junctions in the formation of feather branches: A descriptive ultrastructural study.

PubMed

Alibardi, Lorenzo

2010-08-20

The present study has focused on the distribution and ultrastructure of gap and tight junctions responsible for the formation of the barb/barbule branching in developing feathers using immunocytochemical detection. Apart from desmosomes, both tight and gap junctions are present between differentiating barb/barbule cells and during keratinization. While gap junctions are rare along the perimeter of these cells, tight junctions tend to remain localized in nodes joining barbule cells and between barb cells of the ramus. Occludin and connexin-26 but not connexin-43 have been detected between barb medullary, barb cortical and barbule cells during formation of barbs. Gap junctions are present in supportive cells located in the vicinity of barbule cells and destined to degenerate, but no close junctions are present between supportive and barb/barbule cells. Close junctions mature into penta-laminar junctions that are present between mature barb/barbule cells. Immunolabeling for occludin and Cx26 is rare along these cornified junctions. The junctions allow barb/barbule cells to remain connected until feather-keratin form the mature corneous syncytium that constitutes the barbs. A discussion of the role of gap and tight junctions during feather morphogenesis is presented. 2010 Elsevier GmbH. All rights reserved.

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

PubMed

Martin, Tracey A; Jiang, Wen G

2009-04-01

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

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

PubMed

Lerner, Aaron; Matthias, Torsten

2015-06-01

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

Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

PubMed

Markov, A G; Falchuk, E L; Kruglova, N M; Radloff, J; Amasheh, S

2016-01-01

Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Additive Effects of Rebamipide Plus Proton Pump Inhibitors on the Expression of Tight Junction Proteins in a Rat Model of Gastro-Esophageal Reflux Disease

PubMed Central

Gweon, Tae-Geun; Park, Jong-Hyung; Kim, Byung-Wook; Choi, Yang Kyu; Kim, Joon Sung; Park, Sung Min; Kim, Chang Whan; Kim, Hyung-Gil; Chung, Jun-Won; Incheon

2018-01-01

Background/Aims The aim of this study was to investigate the effects of rebamipide on tight junction proteins in the esophageal mucosa in a rat model of gastroesophageal reflux disease (GERD). Methods GERD was created in rats by tying the proximal stomach. The rats were divided into a control group, a proton pump inhibitor (PPI) group, and a PPI plus rebamipide (PPI+R) group. Pantoprazole (5 mg/kg) was administered intraperitoneally to the PPI and PPI+R groups. An additional dose of rebamipide (100 mg/kg) was administered orally to the PPI+R group. Mucosal erosions, epithelial thickness, and leukocyte infiltration into the esophageal mucosa were measured in isolated esophagi 14 days after the procedure. A Western blot analysis was conducted to measure the expression of claudin-1, -3, and -4. Results The mean surface area of mucosal erosions, epithelial thickness, and leukocyte infiltration were lower in the PPI group and the PPI+R group than in the control group. Western blot analysis revealed that the expression of claudin-3 and -4 was significantly higher in the PPI+R group than in the control group. Conclusions Rebamipide may exert an additive effect in combination with PPI to modify the tight junction proteins of the esophageal mucosa in a rat model of GERD. This treatment might be associated with the relief of GERD symptoms. PMID:29069891

Progressive Hearing Loss in Mice Carrying a Mutation in Usp53

PubMed Central

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

2015-01-01

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

Calcium-Mediated Oxidative Stress: a Common Mechanism in Tight Junction Disruption by Different Types of Cellular Stress

PubMed Central

Gangwar, Ruchika; Meena, Avtar S.; Shukla, Pradeep K.; Nagaraja, Archana S.; Dorniak, Piotr L.; Pallikuth, Sandeep; Waters, Christopher M.; Sood, Anil; Rao, RadhaKrishna

2017-01-01

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction 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 Ca2+ by BAPTA. Knockdown of CaV1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated tight junction disruption and barrier dysfunction. N-acetyl L-cysteine (NAC) and L-nitroarginine methyl ester (L-NAME) blocked stress-induced tight junction disruption and barrier dysfunction. NAC and L-NAME also blocked stress-induced activation of JNK and c-Src. ROS was co-localized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, tight junction disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and tight junction disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca2+, activation of JNK and c-Src, and disruption of tight junction in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, tight junction disruption and protein thiol oxidation in colonic mucosa. This study demonstrates that oxidative stress is a common signal in the mechanism of tight junction disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo. PMID:28057718

Social stress induces neurovascular pathology promoting depression

PubMed Central

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

2017-01-01

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

Tight junction proteins contribute to barrier properties in human pleura.

PubMed

Markov, Alexander G; Voronkova, Maria A; Volgin, George N; Yablonsky, Piotr K; Fromm, Michael; Amasheh, Salah

2011-03-15

The permeability of pleural mesothelium helps to control the volume and composition of the liquid lubricating pleural surfaces. Information on pleural barrier function in health and disease, however, is scarce. Tissue specimens of human pleura were mounted in Ussing chambers for measurement of transmesothelial resistance. Expression of tight junction (TJ) proteins was studied by Western blots and immune fluorescence confocal microscopy. Both visceral and parietal pleura showed barrier properties represented by transmesothelial resistance. Occludin, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7. In tissues from patients with pleural inflammation these tightening claudins were decreased and in visceral pleura claudin-2, a paracellular channel former, became apparent. We report that barrier function in human pleura coincides with expression of claudins known to be key determinants of epithelial barrier properties. In inflamed tissue, claudin expression indicates a reduced barrier function. Copyright © 2010 Elsevier B.V. All rights reserved.

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

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

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

PubMed

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

2012-01-01

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

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

PubMed Central

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

2006-01-01

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

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

PubMed Central

Song, Juhyun; Yoon, So Ra

2017-01-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2011-01-01

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

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

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

W Vallen Graham; A Magis; K Bailey

2011-12-31

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

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

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer.

PubMed

Zihni, Ceniz; Terry, Stephen James

2015-07-01

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

PubMed

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

2017-01-01

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

Effects of Mesalamine Treatment on Gut Barrier Integrity Following Burn Injury

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

Delocalized Claudin-1 promotes metastasis of human osteosarcoma cells

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

Jian, Yuekui; Chen, Changqiong; Li, Bo

2015-10-23

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

Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

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

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

2012-08-31

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

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.

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

PubMed

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

2017-10-01

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

Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression

PubMed Central

Karp, Cristina M.; Tan, Ting Ting; Mathew, Robin; Nelson, Deidre; Mukherjee, Chandreyee; Degenhardt, Kurt; Karantza-Wadsworth, Vassiliki; White, Eileen

2009-01-01

Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial (iBMK) cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity and contact inhibition, while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. PMID:18519669

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

PubMed Central

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

2013-01-01

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

Bile duct epithelial tight junctions and barrier function

PubMed Central

Rao, R.K.; Samak, G.

2013-01-01

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

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

PubMed

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

2010-04-01

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

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

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

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

PubMed

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

2014-01-01

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

The impaired intestinal mucosal immune system by valine deficiency for young grass carp (Ctenopharyngodon idella) is associated with decreasing immune status and regulating tight junction proteins transcript abundance in the intestine.

PubMed

Luo, Jian-Bo; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

2014-09-01

This study investigated the effects of dietary valine on the growth, intestinal immune response, tight junction proteins transcript abundance and gene expression of immune-related signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). Six iso-nitrogenous diets containing graded levels of valine (4.3-19.1 g kg(-)(1) diet) were fed to the fish for 8 weeks. The results showed that percentage weight gain (PWG), feed intake and feed efficiency of fish were the lowest in fish fed the valine-deficient diet (P < 0.05). In addition, valine deficiency decreased lysozyme, acid phosphatase activities and complement 3 content in the intestine (P < 0.05), down-regulated mRNA levels of interleukin 10, transforming growth factor β1, IκBα and target of rapamycin (TOR) (P < 0.05), and up-regulated tumor necrosis factor α, interleukin 8 and nuclear factor κB P65 (NF-κB P65) gene expression (P < 0.05). Additionally, valine deficiency significantly decreased transcript of Occludin, Claudin b, Claudin c, Claudin 3, and ZO-1 (P < 0.05), and improved Claudin 15 expression in the fish intestine (P < 0.05). However, valine did not have a significant effect on expression of Claudin 12 in the intestine of grass carp (P > 0.05). In conclusion, valine deficiency decreased fish growth and intestinal immune status, as well as regulated gene expression of tight junction proteins, NF-κB P65, IκBα and TOR in the fish intestine. Based on the quadratic regression analysis of lysozyme activity or PWG, the dietary valine requirement of young grass carp (268-679 g) were established to be 14.47 g kg(-1) diet (4.82 g 100 g(-1) CP) or 14.00 g kg(-1) diet (4.77 g 100 g(-1) CP), respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Schistosoma japonicum ova maintains epithelial barrier function during experimental colitis.

PubMed

Xia, Chen-Mei; Zhao, Yuan; Jiang, Li; Jiang, Jie; Zhang, Shun-Cai

2011-11-21

To evaluate the impacts of Schistosoma japonicum (S. japonicum) ova on the tight junction barriers in a trinitrobenzenesulfonic acid (TNBS)-induced colitis model. Balb/c mice were randomly divided into three groups: control group; TNBS(+)ova(-) group and TNBS(+)ova(+) group. TNBS was used intracolonic to induce colitis and mice of the TNBS(+)ova(+) group were pre-exposed to S. japonicum ova as a prophylactic intervention. Colon inflammation was quantified using following variables: mouse mortality, weight loss, colon extent and microscopic inflammation score. Serum expression of tumor necrosis factor-α and interferon-γ were assessed to evaluate the systemic inflammatory response. NOD2 and its mRNA were also tested. Bacterial translocations were tested by culturing blood and several tissues. ZO-1 and occludin were chosen as the representations of tight junction proteins. Both the proteins and mRNA were assessed. Ova pre-treatment contributed to the relief of colitis and decreased the mortality of the models. NOD2 expression was significantly downregulated when pretreated with the ova. The TNBS injection caused a significant downregulation of ZO-1 and occludin mRNA together with their proteins in the colon; ova pre-exposure reversed these alterations. Treatment with S. japonicum ova in the colitis model caused lower intestinal bacterial translocation frequency. S. japonicum ova can maintain epithelial barrier function through increasing tight junction proteins, thus causing less exposure of NOD2 to the luminal antigens which may activate a series of inflammatory factors and induce colitis.

Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury

PubMed Central

Chen, Chuanli; Wang, Pei; Su, Qin; Wang, Shiliang; Wang, Fengjun

2012-01-01

Background Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. Methodology/Principal Findings Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. Conclusions/Significance The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury. PMID:22529961

Analytic expression for the giant fieldlike spin torque in spin-filter magnetic tunnel junctions

NASA Astrophysics Data System (ADS)

Tang, Y.-H.; Huang, Z.-W.; Huang, B.-H.

2017-08-01

We propose analytic expressions for fieldlike, T⊥, and spin-transfer, T∥, spin torque components in the spin-filter-based magnetic tunnel junction (SFMTJ), by using the single-band tight-binding model with the nonequilibrium Keldysh formalism. In consideration of multireflection processes between noncollinear magnetization of the spin-filter (SF) barrier and the ferromagnetic (FM) electrode, the central spin-selective SF barrier plays an active role in the striking discovery T⊥≫T∥ , which can be further identified by the unusual barrier thickness dependence of giant T⊥. Our general expressions reveal the sinusoidal angular dependence of both spin torque components, even in the presence of the SF barrier.

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

PubMed

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

2003-06-01

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

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

PubMed

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

2015-01-01

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

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

PubMed

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

2010-08-01

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

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

PubMed

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

2013-01-15

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

The effect of direct-fed microbial supplementation, as an alternative to antibiotics, on growth performance, intestinal immune status and epithelial barrier protein expression in broiler chickens

USDA-ARS?s Scientific Manuscript database

The objective of this study was to investigate the effects of Bacillus subtilis-based probiotic supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine and tight junction (TJ) protein mRNA expression. Day-old broiler chicks (n = 140) were randomly assigne...

The effects of direct-fed microbial supplementation, as alternative to antibiotics, on growth performance, intestinal immune status and epithelial barrier protein expression in broiler chickens

USDA-ARS?s Scientific Manuscript database

This study was conducted to investigate the effects of Bacillus subtilis supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine and tight junction (TJ) protein mRNA expression. Day-old broiler chicks (n = 140) were assigned five dietary treatments: basal...

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

PubMed

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

2017-04-01

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

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Systems Proteomics View of the Endogenous Human Claudin Protein Family

PubMed Central

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

2016-01-01

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

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

PubMed

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

2015-06-01

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

Regulation of tight junction permeability with switch-like speed.

PubMed

Beyenbach, Klaus W

2003-09-01

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

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

PubMed

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

2018-04-01

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

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

PubMed

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

2011-10-01

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

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

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

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

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.

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

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

PubMed

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

2010-12-01

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

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

PubMed

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

2006-08-01

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

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

PubMed

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

2009-08-15

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

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

PubMed

Avaritt, Brittany R; Swaan, Peter W

2014-09-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2011-11-01

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

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

A novel approach to maintain gut mucosal integrity using an oral enzyme supplement.

PubMed

Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Economopoulos, Konstantinos P; Morrison, Sara A; Phupitakphol, Tanit; Tantillo, Tyler J; Gul, Sarah S; Gharedaghi, Mohammad Hadi; Tao, Qingsong; Kaliannan, Kanakaraju; Narisawa, Sonoko; Millán, José L; van der Wilden, Gwendolyn M; Fagenholz, Peter J; Malo, Madhu S; Hodin, Richard A

2014-10-01

To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water. The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.

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

PubMed

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2010-02-12

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

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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2009-09-01

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

Modulation of Intestinal Paracellular Transport by Bacterial Pathogens.

PubMed

Roxas, Jennifer Lising; Viswanathan, V K

2018-03-25

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

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey.

PubMed

Mukendi, Christian; Dean, Nicholas; Lala, Rushil; Smith, Jeramiah; Bronner, Marianne E; Nikitina, Natalya V

2016-01-01

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

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

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels

PubMed Central

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

2017-01-01

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

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

PubMed

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

2016-10-18

Cerebral edema from the disruption of the blood-brain barrier (BBB) after cerebral ischemia is a major cause of morbidity and mortality as well as a common event in patients with stroke. Caveolins (Cavs) are thought to regulate BBB functions. Here, we report for the first time that Cav-1 overexpression (OE) decreased brain edema from BBB disruption following ischemic insult. Edema volumes and Cav-1 expression levels were measured following photothrombosis and middle cerebral artery occlusion (MCAO). Endothelial cells that were transduced with a Cav-1 lentiviral expression vector were transplanted into rats. BBB permeability was quantified with Evans blue extravasation. Edema volume was determined from measures of the extravasation area, brain water content, and average fluorescence intensity after Cy5.5 injections. Tight junction (TJ) protein expression was measured with immunoblotting. Cav-1 expression levels and vasogenic brain edema correlated strongly after ischemic insult. Cav-1 expression and BBB disruption peaked 3 d after the MCAO. In addition, intravenous administration of endothelial cells expressing Cav-1 effectively increased the Cav-1 levels 3 d after the MCAO ischemic insult. Importantly, Cav-1 OE ameliorated the vasogenic edema by inhibiting the degradation of TJ protein expression in the acute phase of ischemic stroke. These results suggested that Cav-1 OE protected the integrity of the BBB mainly by preventing the degradation of TJ proteins in rats. These findings need to be confirmed in a clinical setting in human subjects.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2015-04-27

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

Epithelial Integrity Is Maintained by a Matriptase-Dependent Proteolytic Pathway

PubMed Central

List, Karin; Kosa, Peter; Szabo, Roman; Bey, Alexandra L.; Wang, Chao Becky; Molinolo, Alfredo; Bugge, Thomas H.

2009-01-01

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes. PMID:19717635

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

PubMed

Liddelow, Shane A; Dziegielewska, Katarzyna M; Ek, C Joakim; Habgood, Mark D; Bauer, Hannelore; Bauer, Hans-Christian; Lindsay, Helen; Wakefield, Matthew J; Strazielle, Nathalie; Kratzer, Ingrid; Møllgård, Kjeld; Ghersi-Egea, Jean-François; Saunders, Norman R

2013-01-01

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

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

PubMed

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

2016-01-01

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

Hypoxic Stress and Inflammatory Pain Disrupt Blood-Brain Barrier Tight Junctions: Implications for Drug Delivery to the Central Nervous System.

PubMed

Lochhead, Jeffrey J; Ronaldson, Patrick T; Davis, Thomas P

2017-07-01

A functional blood-brain barrier (BBB) is necessary to maintain central nervous system (CNS) homeostasis. Many diseases affecting the CNS, however, alter the functional integrity of the BBB. It has been shown that various diseases and physiological stressors can impact the BBB's ability to selectively restrict passage of substances from the blood to the brain. Modifications of the BBB's permeability properties can potentially contribute to the pathophysiology of CNS diseases and result in altered brain delivery of therapeutic agents. Hypoxia and/or inflammation are central components of a number of diseases affecting the CNS. A number of studies indicate hypoxia or inflammatory pain increase BBB paracellular permeability, induce changes in the expression and/or localization of tight junction proteins, and affect CNS drug uptake. In this review, we look at what is currently known with regard to BBB disruption following a hypoxic or inflammatory insult in vivo. Potential mechanisms involved in altering tight junction components at the BBB are also discussed. A more detailed understanding of the mediators involved in changing BBB functional integrity in response to hypoxia or inflammatory pain could potentially lead to new treatments for CNS diseases with hypoxic or inflammatory components. Additionally, greater insight into the mechanisms involved in TJ rearrangement at the BBB may lead to novel strategies to pharmacologically increase delivery of drugs to the CNS.

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2016-06-01

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

Supplementation of direct-fed microbial as an alternative to antibiotic on growth performance, intestinal immune status and epithelial barrier integrity in broiler chicken

USDA-ARS?s Scientific Manuscript database

The objective of this study was to investigate the effects of supplementation of broiler diets with Bacillus subtilis-based probiotics on growth performance, feed efficiency, intestinal cytokine and tight junction (TJ) protein mRNA expression. Day-old broiler chicks (n = 140) were randomly assigned...

Dietary Allium hookeri reduces inflammatory response and increases expression of intestinal tight junctions proteins in LPS-induced young broiler chickens

USDA-ARS?s Scientific Manuscript database

We undertook a study to assess the effects of Allium hookeri (AH) root and fermented root on inflammation and intestinal integrity of lipopolysaccharide (LPS)-challenged chickens. Birds were assigned to six groups (n = 25 birds/treatment) and fed with basal diets or basal diets supplemented with AH ...

Ang-(1-7) exerts protective role in blood-brain barrier damage by the balance of TIMP-1/MMP-9.

PubMed

Wu, Jitao; Zhao, Duo; Wu, Shuang; Wang, Dan

2015-02-05

Cerebrovascular disease (CVD) ranks as the top three health risks, specially cerebral ischemia characterized with the damage of blood-brain barrier (BBB). The angiotensin Ang-(1-7) was proven to have a protective effect on cerebrovascular diseases. However, its role on blood-brain barrier and the underlying molecular mechanism remains unclear. In this study, Ang-(1-7) significantly relieved damage of ischemia reperfusion injury on blood-brain barrier in cerebral ischemia reperfusion injury (IRI) rats. Furthermore, its treatment attenuated BBB permeability and brain edema. Similarly, Ang-(1-7) also decreased the barrier permeability of brain endothelial cell line RBE4. Further analysis showed that Ang-(1-7) could effectively restore tight junction protein (claudin-5 and zonula occludens ZO-1) expression levels both in IRI-rats and hypoxia-induced RBE4 cells. Furthermore, Ang-(1-7) stimulation down-regulated hypoxia-induced matrix metalloproteinase-9 (MMP-9) levels, whose silencing with (matrix metalloproteinase-9 hemopexin domain) MMP9-PEX inhibitor significantly increased the expression of claudin-5 and ZO-1. Further mechanism analysis demonstrated that Ang-(1-7) might junction protein levels by tissue inhibitor of metalloproteinase 1 (TIMP1)-MMP9 pathway, because Ang-(1-7) enhanced TIMP1 expression, whose silencing obviously attenuated the inhibitor effect of Ang-(1-7) on MMP-9 levels and decreased Ang-(1-7)-triggered increase in claudin-5 and ZO-1. Together, this study demonstrated a protective role of Ang-(1-7) in IRI-induced blood-brain barrier damage by TIMP1-MMP9-regulated tight junction protein expression. Accordingly, Ang-(1-7) may become a promising therapeutic agent against IRI and its complications. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2016-11-01

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

Changes in barrier health status of the gill for grass carp (Ctenopharyngodon idella) during valine deficiency: Regulation of tight junction protein transcript, antioxidant status and apoptosis-related gene expression.

PubMed

Feng, Lin; Luo, Jian-Bo; Jiang, Wei-Dan; Liu, Yang; Wu, Pei; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Zhang, Yong-An; Zhou, Xiao-Qiu

2015-08-01

This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill (P < 0.05). These effects were partly due to the down-regulation of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and IκB α and the up-regulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and nuclear factor κB P65 (NF-κB P65) (P < 0.05). However, valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills (P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) (P < 0.05). These results may be ascribed to the down-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the up-regulation of Kelch-like-ECH-associated protein 1 (Keap1) (P < 0.05). Additionally, valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions (P < 0.05). These results may be ascribed to the improvement in ROS levels in the fish gill (P < 0.05). Taken together, the results showed that valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gill. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

1997-10-01

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

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

PubMed Central

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

2008-01-01

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

Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes

PubMed Central

Nagaishi, Kanna; Mizue, Yuka; Chikenji, Takako; Otani, Miho; Nakano, Masako; Konari, Naoto; Fujimiya, Mineko

2016-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) have contributed to the improvement of diabetic nephropathy (DN); however, the actual mediator of this effect and its role has not been characterized thoroughly. We investigated the effects of MSC therapy on DN, focusing on the paracrine effect of renal trophic factors, including exosomes secreted by MSCs. MSCs and MSC-conditioned medium (MSC-CM) as renal trophic factors were administered in parallel to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. Both therapies showed approximately equivalent curative effects, as each inhibited the exacerbation of albuminuria. They also suppressed the excessive infiltration of BMDCs into the kidney by regulating the expression of the adhesion molecule ICAM-1. Proinflammatory cytokine expression (e.g., TNF-α) and fibrosis in tubular interstitium were inhibited. TGF-β1 expression was down-regulated and tight junction protein expression (e.g., ZO-1) was maintained, which sequentially suppressed the epithelial-to-mesenchymal transition of tubular epithelial cells (TECs). Exosomes purified from MSC-CM exerted an anti-apoptotic effect and protected tight junction structure in TECs. The increase of glomerular mesangium substrate was inhibited in HFD-diabetic mice. MSC therapy is a promising tool to prevent DN via the paracrine effect of renal trophic factors including exosomes due to its multifactorial action. PMID:27721418

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

PubMed

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

2018-05-17

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

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

PubMed

Jin, Zhe; Ulfendahl, Mats; Järlebark, Leif

2008-01-01

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

Protein kinase C activation modulates reversible increase in cortical blood-brain barrier permeability and tight junction protein expression during hypoxia and posthypoxic reoxygenation.

PubMed

Willis, Colin L; Meske, Diana S; Davis, Thomas P

2010-11-01

Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is a restriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusion phase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, the mechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined the role of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6% O(2)) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluorescein isothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θ distribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538) and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization at endothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKC inhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascular permeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis that nPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.

Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

PubMed

Ouban, Abderrahman; Ahmed, Atif

2015-07-01

Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

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

PubMed Central

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

2010-01-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2018-04-13

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

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

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

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

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

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

PubMed Central

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

2016-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2015-05-01

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

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

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

PubMed

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

2004-09-01

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

Localization of connexin43 in rat kidney.

PubMed

Barajas, L; Liu, L; Tucker, M

1994-09-01

The localization of connexin43 (Cx 43) in rat kidney was investigated by the indirect immunofluorescence technique with polyclonal antisera raised against Cx 43. Cx 43 is a gap junction protein expressed in a variety of tissues. The typically punctuated gap junction immunofluorescence (GJI) was observed in the renal arterial and arteriolar system. In the renal artery the GJI was concentrated in the media. In the juxtamedullary nephrons, the GJI is particularly abundant in the vascular bundles. There is abundant GJI in the extraglomerular mesangium while in the afferent arteriole GJI appears decreased. Abundant GJI was observed in the inner medullary collecting ducts and pelvic epithelium. The localization of Cx 43 immunofluorescence observed in this study is only in partial agreement with the results of ultrastructural investigations on the distribution of gap junctions in the kidney. An extensive tight junctional system has been demonstrated in the collecting duct system. However, gap junctions have been reported to be absent. Further studies to resolve this discrepancy are required.

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice

PubMed Central

Volynets, Valentina; Rings, Andreas; Bárdos, Gyöngyi; Ostaff, Maureen J.; Wehkamp, Jan; Bischoff, Stephan C.

2016-01-01

ABSTRACT The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest. PMID:27583194

Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice.

PubMed

Volynets, Valentina; Rings, Andreas; Bárdos, Gyöngyi; Ostaff, Maureen J; Wehkamp, Jan; Bischoff, Stephan C

2016-01-01

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Claudins and the Kidney Volume 75: Annual Review of Physiology

PubMed Central

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

2013-01-01

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

Immunohistochemical quantification of expression of a tight junction protein, claudin-7, in human lung cancer samples using digital image analysis method.

PubMed

Lu, Zhe; Liu, Yi; Xu, Junfeng; Yin, Hongping; Yuan, Haiying; Gu, Jinjing; Chen, Yan-Hua; Shi, Liyun; Chen, Dan; Xie, Bin

2018-03-01

Tight junction proteins are correlated with cancer development. As the pivotal proteins in epithelial cells, altered expression and distribution of different claudins have been reported in a wide variety of human malignancies. We have previously reported that claudin-7 was strongly expressed in benign bronchial epithelial cells at the cell-cell junction while expression of claudin-7 was either altered with discontinued weak expression or completely absent in lung cancers. Based on these results, we continued working on the expression pattern of claudin-7 and its relationship with lung cancer development. We herein proposed a new Digital Image Classification, Fragmentation index, Morphological analysis (DICFM) method for differentiating the normal lung tissues and lung cancer tissues based on the claudin-7 immunohistochemical staining. Seventy-seven lung cancer samples were obtained from the Second Affiliated Hospital of Zhejiang University and claudin-7 immunohistochemical staining was performed. Based on C++ and Open Source Computer Vision Library (OpenCV, version 2.4.4), the DICFM processing module was developed. Intensity and fragmentation of claudin-7 expression, as well as the morphological parameters of nuclei were calculated. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. Agreement between these computational results and the results obtained by two pathologists was demonstrated. The intensity of claudin-7 expression was significantly decreased while the fragmentation was significantly increased in the lung cancer tissues compared to the normal lung tissues and the intensity was strongly positively associated with the differentiation of lung cancer cells. Moreover, the perimeters of the nuclei of lung cancer cells were significantly greater than that of the normal lung cells, while the parameters of area and circularity revealed no statistical significance. Taken together, our DICFM approach may be applied as an appropriate approach to quantify the immunohistochemical staining of claudin-7 on the cell membrane and claudin-7 may serve as a marker for identification of lung cancer. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Lowering the dietary omega-6: omega-3 does not hinder nonalcoholic fatty-liver disease development in a murine model.

PubMed

Enos, Reilly T; Velázquez, Kandy T; McClellan, Jamie L; Cranford, Taryn L; Walla, Michael D; Murphy, E Angela

2015-05-01

It is hypothesized that a high dietary n-6:n-3 (eg, 10-20:1) is partly responsible for the rise in obesity and related health ailments. However, no tightly controlled studies using high-fat diets differing in the n-6:n-3 have tested this hypothesis. The aim of the study was to determine the role that the dietary n-6:n-3 plays in non-alcoholic fatty-liver disease (NAFLD) and colitis development. We hypothesized that reducing the dietary n-6:n-3 would hinder the development of NAFLD and colitis. Male C57BL/6 J mice were fed high-fat diets, differing in the n-6:n-3 (1:1, 5:1, 10:1, 20:1), for 20 weeks. Gas chromatography-mass spectrometry was used to analyze the hepatic phospholipid arachidonic acid (AA):eicosapentaenoic acid and AA:docosahexaenoic acid. Hepatic metabolism, inflammatory signaling, macrophage polarization, gene expression of inflammatory mediators, oxidative and endoplasmic reticulum stress, and oxidative capacity were assessed as well as colonic inflammatory signaling, and gene expression of inflammatory mediators and tight-junction proteins. Although reducing the dietary n-6:n-3 lowered the hepatic phospholipid AA:eicosapentaenoic acid and AA:docosahexaenoic acid in a dose-dependent manner and mildly influenced inflammatory signaling, it did not significantly attenuate NAFLD development. Furthermore, the onset of NAFLD was not paired to colitis development or changes in tight-junction protein gene expression. In conclusion, reducing the dietary n-6:n-3 did not attenuate NAFLD progression; nor is it likely that colitis, or gut permeability, plays a role in NAFLD initiation in this model. Copyright © 2015 Elsevier Inc. All rights reserved.

Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occludin and reduces fecal calprotectin: a secondary analysis of healthy Wistar Furth rats.

PubMed

Goodrich, Katheryn M; Fundaro, Gabrielle; Griffin, Laura E; Grant, Ar'quette; Hulver, Matthew W; Ponder, Monica A; Neilson, Andrew P

2012-10-01

Animal studies have demonstrated the potential of grape seed extract (GSE) to prevent metabolic syndrome, obesity, and type 2 diabetes. Recently, metabolic endotoxemia induced by bacterial endotoxins produced in the colon has emerged as a possible factor in the etiology of metabolic syndrome. Improving colonic barrier function may control endotoxemia by reducing endotoxin uptake. However, the impact of GSE on colonic barrier integrity and endotoxin uptake has not been evaluated. We performed a secondary analysis of samples collected from a chronic GSE feeding study with pharmacokinetic end points to examine potential modulation of biomarkers of colonic integrity and endotoxin uptake. We hypothesized that a secondary analysis would indicate that chronic GSE administration increases colonic expression of intestinal tight junction proteins and reduces circulating endotoxin levels, even in the absence of an obesity-promoting stimulus. Wistar Furth rats were administered drinking water containing 0.1% GSE for 21 days. Grape seed extract significantly increased the expression of gut junction protein occludin in the proximal colon and reduced fecal levels of the neutrophil protein calprotectin, compared with control. Grape seed extract did not significantly reduce serum or fecal endotoxin levels compared with control, although the variability in serum levels was widely increased by GSE. These data suggest that the improvement of gut barrier integrity and potential modulation of endotoxemia warrant investigation as a possible mechanism by which GSE prevents metabolic syndrome and associated diseases. Further investigation of this mechanism in high-fat feeding metabolic syndrome and obesity models is therefore justified. Copyright © 2012 Elsevier Inc. All rights reserved.

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

DTIC Science & Technology

2004-08-01

AF038388 Vav3 GEF for Rho and Rac Proto-oncogene product NM020505 hPEM -2 GEF for Cdc42 Predominantly expressed in brain AB007884 GEF-H1 GEF for Rac and Rho...fence tions, and desmosomes play a fundamental role in maintain- function). A functional tight junction is crucial to maintain ing the polarized phenotype

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

PubMed

Mehrabadi, Abbas Rezaeian; Korolainen, Minna A; Odero, Gary; Miller, Donald W; Kauppinen, Tiina M

2017-09-01

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulation of gill claudin paralogs by salinity, cortisol and prolactin in Mozambique tilapia (Oreochromis mossambicus).

PubMed

Tipsmark, Christian K; Breves, Jason P; Rabeneck, D Brett; Trubitt, Rebecca T; Lerner, Darren T; Grau, E Gordon

2016-09-01

In euryhaline teleosts, reorganization of gill tight junctions during salinity acclimation involves dynamic expression of specific claudin (Cldn) paralogs. We identified four transcripts encoding Cldn tight junction proteins in the tilapia gill transcriptome: cldn10c, cldn10e, cldn28a and cldn30. A tissue distribution experiment found cldn10c and cldn10e expression levels in the gill to be 100-fold higher than any other tissues examined. cldn28a and cldn30 levels in the gill were 10-fold greater than levels in other tissues. Expression of these genes in Mozambique tilapia was examined during acclimation to fresh water (FW), seawater (SW), and in response to hormone treatments. Transfer of tilapia from FW to SW elevated cldn10c and cldn10e, while cldn28a and cldn30 were stimulated following transfer from SW to FW. In hypophysectomized tilapia transferred to FW, pituitary extirpation induced reduced expression of cldn10c, cldn10e and cldn28a; these effects were mitigated equally by either prolactin or cortisol replacement. In vitro experiments with gill filaments showed that cortisol stimulated expression of all four cldns examined, suggesting a direct action of cortisol in situ. Our data indicate that elevated cldn10c and cldn10e expression is important during acclimation of tilapia to SW possibly by conferring ion specific paracellular permeability. On the other hand, expression of cldn28a and cldn30 appears to contribute to reorganization of branchial epithelium during FW acclimation. Hormone treatment experiments showed that particular FW- and SW-induced cldns are controlled by cortisol and prolactin. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

PubMed

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

2017-08-04

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2014-10-01

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

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

PubMed

Yang, Yi; Kimura-Ohba, Shihoko; Thompson, Jeffrey F; Salayandia, Victor M; Cossé, Melissa; Raz, Limor; Jalal, Fakhreya Y; Rosenberg, Gary A

2018-06-01

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2018-01-29

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

Visceral adipose tissue and leptin increase colonic epithelial tight junction permeability via a RhoA-ROCK-dependent pathway.

PubMed

Le Dréan, Gwenola; Haure-Mirande, Vianney; Ferrier, Laurent; Bonnet, Christian; Hulin, Philippe; de Coppet, Pierre; Segain, Jean-Pierre

2014-03-01

Proinflammatory cytokines produced by immune cells play a central role in the increased intestinal epithelial permeability during inflammation. Expansion of visceral adipose tissue (VAT) is currently considered a consequence of intestinal inflammation. Whether VAT per se plays a role in early modifications of intestinal barrier remains unknown. The aim of this study was to demonstrate the direct role of adipocytes in regulating paracellular permeability of colonic epithelial cells (CECs). We show in adult rats born with intrauterine growth retardation, a model of VAT hypertrophy, and in rats with VAT graft on the colon, that colonic permeability was increased without any inflammation. This effect was associated with altered expression of tight junction (TJ) proteins occludin and ZO-1. In coculture experiments, adipocytes decreased transepithelial resistance (TER) of Caco-2 CECs and induced a disorganization of ZO-1 on TJs. Intraperitoneal administration of leptin to lean rats increased colonic epithelial permeability and altered ZO-1 expression and organization. Treatment of HT29-19A CECs with leptin, but not adiponectin, dose-dependently decreased TER and altered TJ and F-actin cytoskeleton organization through a RhoA-ROCK-dependent pathway. Our data show that adipocytes and leptin directly alter TJ function in CECs and suggest that VAT could impair colonic epithelial barrier.

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

PubMed

Feng, Lin; Gan, Lu; Jiang, Wei-Dan; Wu, Pei; Liu, Yang; Jiang, Jun; Tang, Ling; Kuang, Sheng-Yao; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2017-04-01

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

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

Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.

2010-07-02

The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors ormore » siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.« less

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

PubMed

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

2006-03-01

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

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

PubMed Central

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

2006-01-01

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

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

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

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

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

PubMed Central

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

2002-01-01

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

Frequent loss of claudin-4 expression in dedifferentiated and undifferentiated endometrial carcinomas.

PubMed

Tessier-Cloutier, Basile; Soslow, Robert A; Stewart, Colin J R; Köbel, Martin; Lee, Cheng-Han

2018-04-19

Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UECs) are aggressive endometrial cancers with frequent genomic inactivation of core components of switch/sucrose non-fermentable (SWI/SNF) complex proteins. Claudin-4, an epithelial intercellular tight junction protein, was recently found to be expressed in SWI/SNF-deficient undifferentiated carcinomas but not in SWI/SNF-deficient sarcomas. The aim of this study was to examine claudin-4 expression in UECs/DDECs and other high-grade uterine carcinomas. We examined claudin-4 expression by immunohistochemistry (clone 3E2C1) on tissue microarrays that contained 44 UECs/DDECs (24 SWI/SNF-deficient), 50 carcinosarcomas, 164 grade 3 endometrioid carcinomas, 57 serous carcinomas, and 20 clear cell carcinomas. Tumours with <5% claudin-4 expression were considered to be negative. Nearly all SWI/SNF-deficient, and most SWI/SNF-proficient, UECs/DDECs showed a complete absence of claudin-4 expression in the undifferentiated component, whereas the differentiated component in DDECs showed consistent and diffuse claudin-4 expression. Only one SWI/SNF-deficient DDEC showed focal expression of claudin-4 in the undifferentiated component, as compared with diffuse expression in the corresponding differentiated component. Claudin-4 expression was consistently absent in the sarcomatous component of carcinosarcoma, and it was absent in 24% of grade 3 endometrioid carcinomas and serous carcinomas. Claudin-4 expression can be absent or very focal in a subset of high-grade endometrial carcinomas, and is almost always absent in the undifferentiated components of SWI/SNF-deficient UECs/DDECs, despite the apparent epithelial origin in the case of DDECs. Therefore, claudin-4 expression cannot be used to infer mesenchymal or epithelial tumour origin in the endometrium. The consistent loss or down-regulation of claudin-4, a tight junction protein, in SWI/SNF-deficient UECs/DDECs further supports the undifferentiated nature of these tumours. © 2018 John Wiley & Sons Ltd.

Scutellaria barbata attenuates diabetic retinopathy by preventing retinal inflammation and the decreased expression of tight junction protein

PubMed Central

Mei, Xi-Yu; Zhou, Ling-Yu; Zhang, Tian-Yu; Lu, Bin; Ji, Li-Li

2017-01-01

AIM To observe the attenuation of ethanol extract of Herba Scutellaria barbata (SE) against diabetic retinopathy (DR) and its engaged mechanism. METHODS C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ, 55 mg/kg) for 5 consecutive days to induce diabetes. The diabetic mice were orally given with SE (100, 200 mg/kg) for 1mo at 1mo after STZ injection. Blood-retinal barrier (BRB) breakdown was detected by using Evans blue permeation assay. Real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence staining were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum contents of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. RESULTS SE (100, 200 mg/kg) reversed the breakdown of BRB in STZ-induced diabetic mice. The decreased expression of retinal claudin-1 and claudin-19, which are both tight junction (TJ) proteins, was reversed by SE. SE decreased the increased serum contents and retinal mRNA expression of TNF-α and IL-1β. SE also decreased the increased retinal expression of intercellular cell adhesion molecule-1 (ICAM-1). SE reduced the increased phosphorylation of nuclear factor kappa B (NFκB) p65 and its subsequent nuclear translocation in retinas from STZ-induced diabetic mice. Results of Western blot and retinal immunofluorescence staining of ionized calcium-binding adapter molecule 1 (Iba1) demonstrated that SE abrogated the activation of microglia cells in STZ-induced diabetic mice. CONCLUSION SE attenuates the development of DR by inhibiting retinal inflammation and restoring the decreased expression of TJ proteins including claudin-1 and claudin-19. PMID:28730076

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

PubMed

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

2016-10-11

No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

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

PubMed Central

Dörfel, Max Johannes; Huber, Otmar

2012-01-01

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

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2011-01-01

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

ZO proteins redundantly regulate the transcription factor DbpA/ZONAB.

PubMed

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

2014-08-08

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

Effect of lycopene on the blood-spinal cord barrier after spinal cord injury in mice.

PubMed

Zhang, Qian; Wang, Jianbo; Gu, Zhengsong; Zhang, Qing; Zheng, Hong

2016-09-05

The current study aimed to investigate the effect of lycopene on the blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) in a mouse model. Lycopene inhibited lipid peroxidation and oxidative DNA damage as a highly efficient antioxidant and free radical scavenger. Lycopene (4 mg/kg/d) was administrated immediately following SCI. The permeability of the BSCB and water content in the spinal cord tissue were evaluated. Additionally, levels of expression of tight junction proteins and heme oxygenase-1 (HO-1) were determined with Western blotting. An enzyme-linked immunosorbent assay analysis of spinal cord tissue homogenates was performed 48 h after SCI to evaluate the expression of inflammation-related cytokines. In addition, recovery of motor function was assessed 1 d, 2 d, 5 d, 10 d, and 15 d after SCI using the Basso Mouse Scale to score locomotion. Compared to the group with an untreated SCI, mice with an SCI treated with lycopene had significantly reduced spinal cord tissue water content and BSCB permeability. Furthermore, motor function of mice with an SCI was also greatly improved by lycopene administration. The expression of the proinflammatory factors TNF-α and NF-kB increased markedly 48 h after SCI, and their upregulation was significantly attenuated by lycopene treatment. The expression of molecules that protect tight junctions, zonula occluden-1 and claudin-5, was upregulated by lycopene treatment after SCI. Taken together, these results clearly indicate that lycopene attenuated SCI by promoting repair of the damaged BSCB, so lycopene is a novel and promising treatment for SCI in humans.

Tight junctions of the proximal tubule and their channel proteins.

PubMed

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

2017-08-01

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

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

PubMed Central

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

2011-01-01

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

Subacute stress and chronic stress interact to decrease intestinal barrier function in rats.

PubMed

Lauffer, Adriana; Vanuytsel, Tim; Vanormelingen, Christophe; Vanheel, Hanne; Salim Rasoel, Shadea; Tóth, Joran; Tack, Jan; Fornari, Fernando; Farré, Ricard

2016-01-01

Psychological stress increases intestinal permeability, potentially leading to low-grade inflammation and symptoms in functional gastrointestinal disorders. We assessed the effect of subacute, chronic and combined stress on intestinal barrier function and mast cell density. Male Wistar rats were allocated to four experimental groups (n = 8/group): 1/sham; 2/subacute stress (isolation and limited movement for 24 h); 3/chronic crowding stress for 14 days and 4/combined subacute and chronic stress. Jejunum and colon were collected to measure: transepithelial electrical resistance (TEER; a measure of epithelial barrier function); gene expression of tight junction molecules; mast cell density. Plasma corticosterone concentration was increased in all three stress conditions versus sham, with highest concentrations in the combined stress condition. TEER in the jejunum was decreased in all stress conditions, but was significantly lower in the combined stress condition than in the other groups. TEER in the jejunum correlated negatively with corticosterone concentration. Increased expression of claudin 1, 5 and 8, occludin and zonula occludens 1 mRNAs was detected after subacute stress in the jejunum. In contrast, colonic TEER was decreased only after combined stress, and the expression of tight junction molecules was unaltered. Increased mast cell density was observed in the chronic and combined stress condition in the colon only. In conclusion, our data show that chronic stress sensitizes the gastrointestinal tract to the effects of subacute stress on intestinal barrier function; different underlying cellular and molecular alterations are indicated in the small intestine versus the colon.

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

PubMed

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

2017-04-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

Sukumaran, Sunil K; Prasadarao, Nemani V

2003-11-01

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

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

PubMed

Trubitt, Rebecca T; Rabeneck, D Brett; Bujak, Joanna K; Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

2015-04-01

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena. Copyright © 2014 Elsevier Inc. All rights reserved.

The probiotic mixture IRT5 ameliorates age-dependent colitis in rats.

PubMed

Jeong, Jin-Ju; Woo, Jae-Yeon; Ahn, Young-Tae; Shim, Jae-Hun; Huh, Chul-Sung; Im, Sin-Heog; Han, Myung Joo; Kim, Dong-Hyun

2015-06-01

To investigate the anti-inflammatory effect of probiotics, we orally administered IRT5 (1×10(9)CFU/rat) for 8 weeks to aged (16 months-old) Fischer 344 rats, and measured parameters of colitis. The expression levels of the inflammatory markers' inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were higher in the colons of normal aged rats (18 months-old) than in the colons of normal young rats (6 months-old). Treatment with IRT5 suppressed the age-associated increased expression of iNOS, COX2, TNF-α, and IL-1β, and activation of NF-κB and mitogen-activated protein kinases. In a similar manner, the expression of tight junction proteins in the colon of normal aged rats was suppressed more potently than in normal young rats, and treatment of aged rats with IRT5 decreased the age-dependent suppression of tight junction proteins ZO-1, occludin, and claudin-1. Treatment with IRT5 suppressed age-associated increases in expressions of senescence markers p16 and p53 in the colon of aged rats, but increased age-suppressed expression of SIRT1. However, treatment with IRT5 inhibited age-associated increased myeloperoxidase activity in the colon. In addition, treatment with IRT5 lowered the levels of LPS in intestinal fluid and blood of aged rats, as well as the reduced concentrations of reactive oxygen species, malondialdehyde, and C-reactive protein in the blood. These findings suggest that IRT5 treatment may suppress age-dependent colitis by inhibiting gut microbiota LPS production. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells

PubMed Central

Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

2015-01-01

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell–cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. PMID:25847917

Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

PubMed

Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

2013-01-01

Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

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

PubMed Central

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

2008-01-01

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

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels.

PubMed

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

2016-06-01

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

Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage.

PubMed

Klar, Joakim; Piontek, Jörg; Milatz, Susanne; Tariq, Muhammad; Jameel, Muhammad; Breiderhoff, Tilman; Schuster, Jens; Fatima, Ambrin; Asif, Maria; Sher, Muhammad; Mäbert, Katrin; Fromm, Anja; Baig, Shahid M; Günzel, Dorothee; Dahl, Niklas

2017-07-01

Claudins constitute the major component of tight junctions and regulate paracellular permeability of epithelia. Claudin-10 occurs in two major isoforms that form paracellular channels with ion selectivity. We report on two families segregating an autosomal recessive disorder characterized by generalized anhidrosis, severe heat intolerance and mild kidney failure. All affected individuals carry a rare homozygous missense mutation c.144C>G, p.(N48K) specific for the claudin-10b isoform. Immunostaining of sweat glands from patients suggested that the disease is associated with reduced levels of claudin-10b in the plasma membranes and in canaliculi of the secretory portion. Expression of claudin-10b N48K in a 3D cell model of sweat secretion indicated perturbed paracellular Na+ transport. Analysis of paracellular permeability revealed that claudin-10b N48K maintained cation over anion selectivity but with a reduced general ion conductance. Furthermore, freeze fracture electron microscopy showed that claudin-10b N48K was associated with impaired tight junction strand formation and altered cis-oligomer formation. These data suggest that claudin-10b N48K causes anhidrosis and our findings are consistent with a combined effect from perturbed TJ function and increased degradation of claudin-10b N48K in the sweat glands. Furthermore, affected individuals present with Mg2+ retention, secondary hyperparathyroidism and mild kidney failure that suggest a disturbed reabsorption of cations in the kidneys. These renal-derived features recapitulate several phenotypic aspects detected in mice with kidney specific loss of both claudin-10 isoforms. Our study adds to the spectrum of phenotypes caused by tight junction proteins and demonstrates a pivotal role for claudin-10b in maintaining paracellular Na+ permeability for sweat production and kidney function.

Curcumin improves intestinal barrier function: modulation of intracellular signaling, and organization of tight junctions.

PubMed

Wang, Jing; Ghosh, Siddhartha S; Ghosh, Shobha

2017-04-01

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as type 2 diabetes and atherosclerosis) has shifted the focus from high-fat high-cholesterol containing Western-type diet (WD)-induced changes in gut microbiota per se to release of gut bacteria-derived products (e.g., LPS) into circulation due to intestinal barrier dysfunction as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that oral supplementation with curcumin attenuates WD-induced development of type 2 diabetes and atherosclerosis. Poor bioavailability of curcumin has precluded the establishment of a causal relationship between oral supplementation and it is in vivo effects. We hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the intestinal barrier function. The objective of the present study was to delineate the underlying mechanisms. The human IEC lines Caco-2 and HT-29 were used for these studies and modulation of direct as well as indirect effects of LPS on intracellular signaling as well as tight junctions were examined. Pretreatment with curcumin significantly attenuated LPS-induced secretion of master cytokine IL-1β from IECs and macrophages. Furthermore, curcumin also reduced IL-1β-induced activation of p38 MAPK in IECs and subsequent increase in expression of myosin light chain kinase involved in the phosphorylation of tight junction proteins and ensuing disruption of their normal arrangement. The major site of action of curcumin is, therefore, likely the IECs and the intestinal barrier, and by reducing intestinal barrier dysfunction, curcumin modulates chronic inflammatory diseases despite poor bioavailability. Copyright © 2017 the American Physiological Society.

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

PubMed Central

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

2015-01-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti–IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti–IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti–IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.-E., Bodge, C. A., Cummings, E., Lim, Y.-P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus. PMID:25609424

Transdermal anti-nuclear kappaB siRNA therapy for atopic dermatitis using a combination of two kinds of functional oligopeptide.

PubMed

Ibaraki, Hisako; Kanazawa, Takanori; Takashima, Yuuki; Okada, Hiroaki; Seta, Yasuo

2018-05-05

Nucleic acid-based targeting of nuclear factor kappaB (NF-κB) is gaining attention as a treatment option for skin diseases like atopic dermatitis (AD). Transdermal administration improves patient quality of life because of non-invasive; however, siRNA delivery into the skin can be challenging owing to the barrier of tight junctions in the granular layer. Therefore, we aimed to develop a delivery system of siRNA for topical skin application using functional peptides. We previously reported that combined treatment with a cytoplasm-responsive stearylated-arginine-rich peptide (STR-CH 2 R 4 H 2 C) and a tight junction opening peptide (AT1002) showed high siRNA permeability in the skin of AD-induced and normal mice. Here, we used murine macrophage RAW264.7 cells to examine siRNA permeation and the therapeutic effect of anti-NF-κB (RelA) siRNA (siRelA) complexed with STR-CH 2 R 4 H 2 C and AT1002 for AD-induced mice. We showed that significantly higher siRNA cellular uptake occurs after this treatment as well as decreased TNF-α and IL-6 expression. Additionally, we showed that effective siRNA transdermal delivery occurs with the suppression of the tight junction protein ZO-1. Moreover, topical skin application of siRelA with STR-CH 2 R 4 H 2 C and AT1002 improved AD-like symptoms in model mice. Thus, the combined treatment of STR-CH 2 R 4 H 2 C and AT1002 could serve as an effective transdermal siRNA therapeutic system for AD. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

1995-09-01

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

Dopamine enhances duodenal epithelial permeability via the dopamine D5 receptor in rodent.

PubMed

Feng, X-Y; Zhang, D-N; Wang, Y-A; Fan, R-F; Hong, F; Zhang, Y; Li, Y; Zhu, J-X

2017-05-01

The intestinal barrier is made up of epithelial cells and intercellular junctional complexes to regulate epithelial ion transport and permeability. Dopamine (DA) is able to promote duodenal epithelial ion transport through D1-like receptors, which includes subtypes of D 1 (D 1 R) and D 5 (D 5 R), but whether D1-like receptors influence the duodenal permeability is unclear. FITC-dextran permeability, short-circuit current (I SC ), Western blot, immunohistochemistry and ELISA were used in human D 5 R transgenic mice and hyperendogenous enteric DA (HEnD) rats in this study. Dopamine induced a downward deflection in I SC and an increase in FITC-dextran permeability of control rat duodenum, which were inhibited by the D1-like receptor antagonist, SCH-23390. However, DA decreased duodenal transepithelial resistance (TER), an effect also reversed by SCH-23390. A strong immunofluorescence signal for D 5 R, but not D 1 R, was observed in the duodenum of control rat. In human D 5 R knock-in transgenic mice, duodenal mucosa displayed an increased basal I SC with high FITC-dextran permeability and decreased TER with a lowered expression of tight junction proteins, suggesting attenuated duodenal barrier function in these transgenic mice. D 5 R knock-down transgenic mice manifested a decreased basal I SC with lowered FITC-dextran permeability. Moreover, an increased FITC-dextran permeability combined with decreased TER and tight junction protein expression in duodenal mucosa were also observed in HEnD rats. This study demonstrates, for the first time, that DA enhances duodenal permeability of control rat via D 5 R, which provides new experimental and theoretical evidence for the influence of DA on duodenal epithelial barrier function. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Tonic regulation of vascular permeability

PubMed Central

Curry, Fitz-Roy E.; Adamson, Roger H.

2014-01-01

Our major theme is that the layered structure of the endothelial barrier requires continuous activation of signaling pathways regulated by S1P and intracellular cAMP. These pathways modulate the adherens junction, continuity of tight junction strands, and the balance of synthesis and degradation of glycocalyx components. We evaluate recent evidence that baseline permeability is maintained by constant activity of mechanisms involving the small GTPases Rap1 and Rac1. In the basal state, the barrier is compromised when activities of the small GTPases are reduced by low S1P supply or delivery. With inflammatory stimulus, increased permeability can be understood in part as the action of signaling to reduce Rap1 and Rac1 activation. With the hypothesis that microvessel permeability and selectivity under both normal and inflammatory conditions are regulated by mechanisms that are continuously active it follows that when S1P or intracellular cAMP are elevated at the time of inflammatory stimulus, they can buffer changes induced by inflammatory agents and maintain normal barrier stability. When endothelium is exposed to inflammatory conditions and subsequently exposed to elevated S1P or intracellular cAMP, the same processes restore the functional barrier by first reestablishing the adherens junction, then modulating tight junctions and glycocalyx. In more extreme inflammatory conditions, loss of the inhibitory actions of Rac1 dependent mechanisms may promote expression of more inflammatory endothelial phenotypes by contributing to the up-regulation of RhoA dependent contractile mechanisms and the sustained loss of surface glycocalyx allowing access of inflammatory cells to the endothelium. PMID:23374222

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

PubMed

Yin, Qingqiao; Xia, Yuanyu; Wang, Guan

2016-09-02

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

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

PubMed Central

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

2003-01-01

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

Perfect transmission at oblique incidence by trigonal warping in graphene P-N junctions

NASA Astrophysics Data System (ADS)

Zhang, Shu-Hui; Yang, Wen

2018-01-01

We develop an analytical mode-matching technique for the tight-binding model to describe electron transport across graphene P-N junctions. This method shares the simplicity of the conventional mode-matching technique for the low-energy continuum model and the accuracy of the tight-binding model over a wide range of energies. It further reveals an interesting phenomenon on a sharp P-N junction: the disappearance of the well-known Klein tunneling (i.e., perfect transmission) at normal incidence and the appearance of perfect transmission at oblique incidence due to trigonal warping at energies beyond the linear Dirac regime. We show that this phenomenon arises from the conservation of a generalized pseudospin in the tight-binding model. We expect this effect to be experimentally observable in graphene and other Dirac fermions systems, such as the surface of three-dimensional topological insulators.

Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

PubMed Central

Bao, Jialing; Yura, Renee E.; Matters, Gail L.; Bradley, S. Gaylen; Shi, Pan; Tian, Fang

2013-01-01

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation. PMID:23804454

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

PubMed

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

2007-01-26

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

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

PubMed Central

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

2007-01-01

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

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

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

2018-01-01

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

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

PubMed

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

2017-03-01

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

The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.

PubMed

Li, X; Lynn, B D; Nagy, J I

2012-01-01

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

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

PubMed

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

2017-06-01

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

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

PubMed

Lechuga, Susana; Ivanov, Andrei I

2017-07-01

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

Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature.

PubMed

Alvarez, Jorge I; Teale, Judy M

2007-09-12

The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption was explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) were evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity.

Effects of topical steroids on tight junction proteins and spongiosis in esophageal epithelia of patients with eosinophilic esophagitis.

PubMed

Katzka, David A; Tadi, Ravikanth; Smyrk, Thomas C; Katarya, Eesha; Sharma, Anamay; Geno, Deborah M; Camilleri, Michael; Iyer, Prasad G; Alexander, Jeffrey A; Buttar, Navtej S

2014-11-01

The allergic response associated with eosinophilic esophagitis (EoE) occurs when food antigens permeate tight junction-mediated epithelial dilated intercellular spaces. We assessed whether levels of tight junction proteins correlate with the dilation of intercellular spaces (spongiosis) and the effects of topical steroids on these parameters. We assessed esophageal biopsy samples from 10 patients with active EoE treated with topical fluticasone, 10 untreated patients, and 10 patients without esophageal disease (controls) for degree of spongiosis. Immunohistochemical assays were used to determine the levels of the tight junction proteins filaggrin, zonula occludens (ZO)-1, ZO-2, ZO-3, and claudin-1. Histology and immunohistochemistry results were assessed blindly, with levels of tight junction proteins and degree of spongiosis rated on scales of 0 to 3. The mean degrees of spongiosis in untreated and treated patients with EoE were 1.3 and 0.4, respectively (P = .016). Esophageal epithelia did not stain significantly for ZO-1 or ZO-2. Filaggrin was observed in a predominant cytoplasmic pattern, compared with the cytoplasmic and membranous patterns of ZO-3 and claudin-1. In biopsy specimens from patients with active EoE, the mean staining intensities for filaggrin, ZO-3, and claudin-1 were 1.6, 1.4, and 0.7, respectively. In biopsy specimens from patients treated with fluticasone, levels of filaggrin, ZO-3, and claudin-1 were 2.8 (P = .002 compared with untreated patients), 1.7 (P = .46 compared with untreated patients), and 1.3 (P = .25 compared with untreated patients), respectively. The correlation between the level of filaggrin and the degree of spongiosis was r = 0.23, and between ZO-3 staining and the degree of spongiosis was r = .016 (P = .001 for filaggrin vs ZO-3 staining). Filaggrin, ZO-3, and claudin-1 (but not ZO-1 or ZO-2) are detected in the esophageal mucosa of patients with EoE treated with steroids and individuals without esophageal disease. Without treatment, spongiosis increases, corresponding with reduced levels of filaggrin, ZO-3, and claudin-1. Loss of tight junction regulators and dilation of intercellular spaces appear to be involved in the pathophysiology of EoE and could be targets for treatment. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Reduced E-cadherin expression is associated with abdominal pain and symptom duration in a study of alternating and diarrhea predominant IBS.

PubMed

Wilcz-Villega, E; McClean, S; O'Sullivan, M

2014-03-01

Increased intestinal permeability and altered expression of tight junction (TJ) proteins may be implicated in the pathogenesis of irritable bowel syndrome (IBS). This study aimed to investigate the expression of adherens junction (AJ) protein E-cadherin and TJ proteins zonula occludens (ZO)-1 and claudin (CLD)-1 and associations with IBS symptoms. Junctional proteins were immunostained in cecal biopsy tissue of Rome II IBS patients (n = 34) comprising both alternating (IBS-A) and diarrhea predominant (IBS-D) subtypes, and controls (n = 12). IBS symptom duration, abdominal pain severity and stool frequency were assessed for IBS patients. Protein expression was determined by immunofluorescence. E-cadherin and ZO-1 protein expression was significantly lower (p = 0.03 and p = 0.016, respectively) in the cecal surface epithelium of the IBS group comprising both IBS-A and IBS-D subtypes. CLD-1 expression was not significantly altered compared with controls. On subtype analysis, ZO-1 expression was significantly reduced in both IBS-A and IBS-D compared with controls, whereas E-cadherin was reduced only in IBS-A. Lower E-cadherin expression was associated with longer symptoms duration specifically in IBS-A patients (rs = -0.76, p = 0.004). Reduced E-cadherin associated with abdominal pain severity in the overall IBS group (rs = -0.36, p = 0.041), but this association was unrelated to IBS subtype. E-cadherin protein expression in the cecum was significantly lower in IBS-A compared with controls and associated with longstanding symptoms. E-cadherin was further associated with abdominal pain severity in the IBS group overall, but unrelated to IBS subtype. Altered E-cadherin expression may provide novel insights into mechanisms underlying intestinal barrier dysfunction in IBS. © 2013 John Wiley & Sons Ltd.

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

PubMed

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

2017-01-02

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

Voltage-gated sodium channels in taste bud cells.

PubMed

Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

2009-03-12

Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

PubMed

Looi, K; Buckley, A G; Rigby, P J; Garratt, L W; Iosifidis, T; Zosky, G R; Larcombe, A N; Lannigan, F J; Ling, K-M; Martinovich, K M; Kicic-Starcevich, E; Shaw, N C; Sutanto, E N; Knight, D A; Kicic, A; Stick, S M

2018-05-01

Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; R T ) and permeability to fluorescent dextran. Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma. © 2018 John Wiley & Sons Ltd.

Cyclooxygenase-2 Deficiency Leads to Intestinal Barrier Dysfunction and Increased Mortality During Polymicrobial Sepsis 1

PubMed Central

Fredenburgh, Laura E.; Velandia, Margarita M. Suarez; Ma, Jun; Olszak, Torsten; Cernadas, Manuela; Englert, Joshua A.; Chung, Su Wol; Liu, Xiaoli; Begay, Cynthia; Padera, Robert F.; Blumberg, Richard S.; Walsh, Stephen R.; Baron, Rebecca M.; Perrella, Mark A.

2011-01-01

Sepsis remains the leading cause of death in critically ill patients despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase-2 (COX-2) is highly upregulated in the intestine during sepsis and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2−/− and COX-2+/+ BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD2, or vehicle and stimulated with cytokines. COX-2−/− mice developed exaggerated bacteremia and increased mortality compared with COX-2+/+ mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD2 attenuated cytokine-induced hyperpermeability and ZO-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis. PMID:21967897

Alteration of Tight Junction Protein Expression in Dahl Salt-Sensitive Rat Kidney.

PubMed

Jo, Chor Ho; Kim, Sua; Oh, Il Hwan; Park, Joon-Sung; Kim, Gheun-Ho

2017-01-01

Altered pressure natriuresis is an important mechanism of hypertension, but it remains elusive at the molecular level. We hypothesized that in the kidney, tight junctions (TJs) may have a role in pressure natriuresis because paracellular NaCl transport affects interstitial hydrostatic pressure. To assess the association of salt-sensitive hypertension with altered renal TJ protein expression, Dahl salt-sensitive (SS) and salt-resistant (SR) rats were put on an 8% NaCl-containing rodent diet for 4 weeks. Systolic blood pressure (SBP) and urine NaCl excretion were measured weekly, and kidneys were harvested for immunoblotting and quantitative PCR analysis at the end of the animal experiments. SBP was significantly higher in SS rats than in SR rats during the first to fourth weeks of the animal experiments. During the first and second week, urinary NaCl excretion was significantly lower in SS rats as compared with SR rats. However, the difference between the two groups vanished at the third and fourth weeks. In the kidney, claudin-4 protein and mRNA were significantly increased in SS rats as compared with SR rats. On the other hand, occludin protein and mRNA were significantly decreased in SS rats as compared with SR rats. The expression of claudin-2, claudin-7, and claudin-8 did not vary significantly between the two groups. In SS rats, SS hypertension was associated with differential changes in renal TJ protein expression. Both upregulation of claudin-4 and downregulation of occludin might increase paracellular NaCl transport in the kidney, resulting in impaired pressure natriuresis in SS rats. © 2017 The Author(s). Published by S. Karger AG, Basel.

Hypersensitivity to acid is associated with impaired esophageal mucosal integrity in patients with gastroesophageal reflux disease with and without esophagitis.

PubMed

Weijenborg, Pim W; Smout, André J P M; Verseijden, Caroline; van Veen, Henk A; Verheij, Joanne; de Jonge, Wouter J; Bredenoord, Albert J

2014-08-01

Increased esophageal sensitivity and impaired mucosal integrity have both been described in patients with gastroesophageal reflux disease, but the relationship between hypersensitivity and mucosal integrity is unclear. The aim of the present study was to investigate acid sensitivity in patients with erosive and nonerosive reflux disease and control subjects to determine the relation with functional esophageal mucosal integrity changes as well as to investigate cellular mechanisms of impaired mucosal integrity in these patients. In this prospective experimental study, 12 patients with nonerosive reflux disease, 12 patients with esophagitis grade A or B, and 11 healthy control subjects underwent an acid perfusion test and upper endoscopy. Mucosal integrity was measured during endoscopy by electrical tissue impedance spectroscopy and biopsy specimens were analyzed in Ussing chambers for transepithelial electrical resistance, transepithelial permeability and gene expression of tight junction proteins and filaggrin. Patients with nonerosive reflux disease and esophagitis were more sensitive to acid perfusion compared with control subjects, having a shorter time to perception of heartburn and higher perceived intensity of heartburn. In reflux patients, enhanced acid sensitivity was associated with impairment of in vivo and vitro esophageal mucosal integrity. Mucosal integrity was significantly impaired in patients with esophagitis, displaying higher transepithelial permeability and lower extracellular impedance. Although no significant differences in the expression of tight junction proteins were found in biopsies among patient groups, mucosal integrity parameters in reflux patients correlated negatively with the expression of filaggrin. In conclusion, sensitivity to acid is enhanced in patients with gastroesophageal reflux disease, irrespective of the presence of erosions, and is associated with impaired esophageal mucosal integrity. Mucosal integrity of the esophagus is associated with the expression of filaggrin. Copyright © 2014 the American Physiological Society.

HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.

PubMed

Wang, Hua; Sun, Rui-Ting; Li, Yang; Yang, Yue-Feng; Xiao, Feng-Jun; Zhang, Yi-Kun; Wang, Shao-Xia; Sun, Hui-Yan; Zhang, Qun-Wei; Wu, Chu-Tse; Wang, Li-Sheng

2015-01-01

Effective therapeutic strategies to address intestinal complications after radiation exposure are currently lacking. Mesenchymal stem cells (MSCs), which display the ability to repair the injured intestine, have been considered as delivery vehicles for repair genes. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF)-gene-modified MSCs on radiation-induced intestinal injury (RIII). Female 6- to 8-week-old mice were radiated locally at the abdomen with a single 13-Gy dose of radiation and then treated with saline control, Ad-HGF or Ad-Null-modified MSCs therapy. The transient engraftment of human MSCs was detected via real-time PCR and immunostaining. The therapeutic effects of non- and HGF-modified MSCs were evaluated via FACS to determine the lymphocyte immunophenotypes; via ELISA to measure cytokine expression; via immunostaining to determine tight junction protein expression; via PCNA staining to examine intestinal epithelial cell proliferation; and via TUNEL staining to detect intestinal epithelial cell apoptosis. The histopathological recovery of the radiation-injured intestine was significantly enhanced following non- or HGF-modified MSCs treatment. Importantly, the radiation-induced immunophenotypic disorders of the mesenteric lymph nodes and Peyer's patches were attenuated in both MSCs-treated groups. Treatment with HGF-modified MSCs reduced the expression and secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), increased the expression of the anti-inflammatory cytokine IL-10 and the tight junction protein ZO-1, and promoted the proliferation and reduced the apoptosis of intestinal epithelial cells. Treatment of RIII with HGF-gene-modified MSCs reduces local inflammation and promotes the recovery of small intestinal histopathology in a mouse model. These findings might provide an effective therapeutic strategy for RIII.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

PubMed

Úbeda, María; Lario, Margaret; Muñoz, Leticia; Borrero, María-José; Rodríguez-Serrano, Macarena; Sánchez-Díaz, Ana-María; Del Campo, Rosa; Lledó, Lourdes; Pastor, Óscar; García-Bermejo, Laura; Díaz, David; Álvarez-Mon, Melchor; Albillos, Agustín

2016-05-01

In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Green tea polyphenols alleviate early BBB damage during experimental focal cerebral ischemia through regulating tight junctions and PKCalpha signaling.

PubMed

Liu, Xiaobai; Wang, Zhenhua; Wang, Ping; Yu, Bo; Liu, Yunhui; Xue, Yixue

2013-07-21

It has been supposed that green tea polyphenols (GTPs) have neuroprotective effects on brain damage after brain ischemia in animal experiments. Little is known regarding GTPs' protective effects against the blood-brain barrier (BBB) disruption after ischemic stroke. We investigated the effects of GTPs on the expression of claudin-5, occludin, and ZO-1, and the corresponding cellular mechanisms involved in the early stage of cerebral ischemia. Male Wistar rats were subjected to a middle cerebral artery occlusion (MCAO) for 0, 30, 60, and 120 min. GTPs (400 mg/kg/day) or vehicle was administered by intragastric gavage twice a day for 30 days prior to MCAO. At different time points, the expression of claudin-5, occludin, ZO-1, and PKCα signaling pathway in microvessel fragments of cerebral ischemic tissue were evaluated. GTPs reduced BBB permeability at 60 min and 120 min after ischemia as compared with the vehicle group. Transmission electron microscopy also revealed that GTPs could reverse the opening of tight junction (TJ) barrier at 60 min and 120 min after MACO. The decreased mRNA and protein expression levels of claudin-5, occludin, and ZO-1 in microvessel fragments of cerebral ischemic tissue were significantly prevented by treatment with GTPs at the same time points after ischemia in rats. Furthermore, GTPs could attenuate the increase in the expression levels of PKCα mRNA and protein caused by cerebral ischemia. These results demonstrate that GTPs may act as a potential neuroprotective agent against BBB damage at the early stage of focal cerebral ischemia through the regulation of TJ and PKCα signaling.

Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats.

PubMed

Li, Jiajia; Zhang, Rong; Wang, Caixia; Wang, Xin; Xu, Man; Ma, Jingxue; Shang, Qingli

2018-03-30

Choroidal neovascularization (CNV) is a common vision-threatening complication associated with many  fundus diseases. The retinal pigment epithelial (RPE) cell junction barrier has critical functions in preventing CNV, and oxidative stress can cause compromise of barrier integrity and induce angiogenesis. Rap1, a small guanosine triphosphatase (GTPase), is involved in regulating endothelial and epithelial cell junctions. In this work, we explored the function and mechanism of Rap1 in CNV in vivo. A laser-induced rat CNV model was developed. Rap1 was activated through intravitreal injection of the Rap1 activator 8CPT-2'-O-Me-cAMP (8CPT). At 14 days after laser treatment, CNV size in RPE/choroid flat mounts was measured by fluorescein isothiocyanate-dextran staining. Expression of vascular endothelial growth factor (VEGF) and cell junction proteins in RPE/choroid tissues were analyzed by western blots and quantitative real-time PCR assays. Reactive oxygen species (ROS) in RPE cells were detectedbydichloro-dihydro-fluorescein diacetate assays. The antioxidant apocynin was intraperitoneally injected into rats. Activating Rap1 by 8CPT significantly reduced CNV size and VEGF expression in the rat CNV model. Rap1 activation enhanced protein and mRNA levels of ZO-1 and occludin, two tight junction proteins in the RPE barrier. In addition, reducing ROS generation by injection of apocynin, a NADPH oxidase inhibitor, inhibited CNV formation. Rap1 activation reduced ROS generation and expression of NADPH oxidase 4. Rap1 activation inhibits CNV through regulating barrier integrity and ROS generation of RPE in vivo, and selectively activating Rap1 may be a way to reduce vision loss from CNV.

Fecal microbiota transplantation prevents hepatic encephalopathy in rats with carbon tetrachloride-induced acute hepatic dysfunction.

PubMed

Wang, Wei-Wei; Zhang, Yu; Huang, Xiao-Bing; You, Nan; Zheng, Lu; Li, Jing

2017-10-14

To investigate whether fecal microbiota transplantation (FMT) prevents hepatic encephalopathy (HE) in rats with carbon tetrachloride (CCl 4 )-induced acute hepatic dysfunction. A rat model of HE was established with CCl 4 . Rat behaviors and spatial learning capability were observed, and hepatic necrosis, intestinal mucosal barrier, serum ammonia levels and intestinal permeability were determined in HE rats receiving FMT treatment. Furthermore, the expression of tight junction proteins (Claudin-1, Claudin-6 and Occludin), Toll-like receptor (TLR) 4/TLR9, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α was examined. FMT improved rat behaviors, HE grade and spatial learning capability. Moreover, FMT prevented hepatic necrosis and intestinal mucosal barrier damage, leading to hepatic clearance of serum ammonia levels and reduced intestinal permeability. The expression of TLR4 and TLR9, two potent mediators of inflammatory response, was significantly downregulated in the liver of rats treated with FMT. Consistently, circulating pro-inflammatory factors such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were remarkably decreased, indicating that FMT is able to limit systemic inflammation by decreasing the expression of TLR4 and TLR9. Importantly, HE-induced loss of tight junction proteins (Claudin-1, Claudin-6 and Occludin) was restored in intestinal tissues of rats receiving FMT treatment. FMT enables protective effects in HE rats, and it improves the cognitive function and reduces the liver function indexes. FMT may cure HE by altering the intestinal permeability and improving the TLR response of the liver.

Cardiotonic steroid ouabain stimulates expression of blood-testis barrier proteins claudin-1 and -11 and formation of tight junctions in Sertoli cells.

PubMed

Dietze, Raimund; Shihan, Mazen; Stammler, Angelika; Konrad, Lutz; Scheiner-Bobis, Georgios

2015-04-15

The interaction of ouabain with the sodium pump induces signalling cascades resembling those triggered by hormone/receptor interactions. In the rat Sertoli cell line 93RS2, ouabain at low concentrations stimulates the c-Src/c-Raf/Erk1/2 signalling cascade via its interaction with the α4 isoform of the sodium pump expressed in these cells, leading to the activation of the transcription factor CREB. As a result of this signalling sequence, ouabain stimulates expression of claudin-1 and claudin-11, which are also controlled by a CRE promoter. Both of these proteins are known to be essential constituents of tight junctions (TJ) between Sertoli cells, and as a result of the ouabain-induced signalling TJ formation between neighbouring Sertoli cells is significantly enhanced by the steroid. Thus, ouabain-treated cell monolayers display higher transepithelial resistance and reduced free diffusion of FITC-coupled dextran in tracer diffusion assays. Taking into consideration that the formation of TJ is indispensable for the maintenance of the blood-testis barrier (BTB) and therefore for male fertility, the actions of ouabain described here and the fact that this and other related cardiotonic steroids (CTS) are produced endogenously suggest a direct influence of ouabain/sodium pump interactions on the maintenance of the BTB and thereby an effect on male fertility. Since claudin-1 and claudin-11 are also present in other blood-tissue barriers, one can speculate that ouabain and perhaps other CTS influence the dynamics of these barriers as well. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Safety evaluation of a human chimeric monoclonal antibody that recognizes the extracellular loop domain of claudin-2.

PubMed

Hashimoto, Yosuke; Hata, Tomoyuki; Tada, Minoru; Iida, Manami; Watari, Akihiro; Okada, Yoshiaki; Doi, Takefumi; Kuniyasu, Hiroki; Yagi, Kiyohito; Kondoh, Masuo

2018-05-30

Claudin-2 (CLDN-2), a pore-forming tight junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown. We recently generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Here, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human-rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcγ receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

PubMed

Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

2015-11-01

See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions.

PubMed

Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

2014-01-01

Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.

Dietary phenylalanine-improved intestinal barrier health in young grass carp (Ctenopharyngodon idella) is associated with increased immune status and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes and related signalling molecules.

PubMed

Feng, Lin; Li, Wen; Liu, Yang; Jiang, Wei-Dan; Kuang, Sheng-Yao; Jiang, Jun; Tang, Ling; Wu, Pei; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2015-08-01

The present work evaluated the effects of dietary phenylalanine (Phe) on the intestinal immune response, tight junction proteins transcript abundance, and the gene expression of immune- and antioxidant-related signalling molecules in the intestine. In addition, the dietary Phe (and Phe + Tyr) requirement of young grass carp (Ctenopharyngodon idella) was also estimated. Fish were fed fish meal-casein-gelatin based diets (302.3 g crude protein kg(-1)) containing 3.4 (basal diet), 6.1, 9.1, 11.5, 14.0 and 16.8 g Phe kg(-1) with a fixed amount of 10.7 g tyrosine kg(-1) for 8 weeks. The results showed that Phe deficiency or excess Phe reduced the lysozyme and acid phosphatase activities and complement C 3 content in the intestine (P < 0.05). Moreover, zonula occludens-1 (ZO-1), occludin and claudin c mRNA levels were highest in the fish fed the diet containing 11.5 g Phe kg(-1) (P < 0.05). However, claudin 12 and claudin b mRNA levels were not significantly affected by dietary Phe (P > 0.05). Gene expression of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), target of rapamycin (TOR) and inhibitor of nuclear factor κBα (IκBα) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) increased as dietary Phe increased up to 6.1, 9.1, 11.5 and 14.0 g kg(-1), respectively (P < 0.05). However, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear factor-κB p65 (NF-κB p65) mRNA levels showed opposite tendencies. In addition, the mRNA level of superoxide dismutase (SOD) was significantly lower in the intestinal tissue of the group fed a diet with Phe levels of 16.8 g kg(-1) than in those of other groups (P < 0.05). The expression of NF-E2-related factor 2 (Nrf2) gene was increased as dietary Phe increased up to 9.1 g kg(-1) (P < 0.05). In conclusion, Phe improved intestinal immune status, and regulated gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, IκBα, TOR, and Nrf2 in the fish intestine. Based on the quadratic regression analysis of lysozyme activity at a 95% maximum, the dietary Phe requirement of young grass carp (256-629 g) was estimated to be 8.31 g kg(-1), corresponding to 2.75 g 100 g(-1) protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Safety concerns over the use of intestinal permeation enhancers: A mini-review.

PubMed

McCartney, Fiona; Gleeson, John P; Brayden, David J

2016-01-01

Intestinal permeation enhancers (PEs) are key components in ∼12 oral peptide formulations in clinical trials for a range of molecules, primarily insulin and glucagon-like-peptide 1 (GLP-1) analogs. The main PEs comprise medium chain fatty acid-based systems (sodium caprate, sodium caprylate, and N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC)), bile salts, acyl carnitines, and EDTA. Their mechanism of action is complex with subtle differences between the different molecules. With the exception of SNAC and EDTA, most PEs fluidize the plasma membrane causing plasma membrane perturbation, as well as enzymatic and intracellular mediator changes that lead to alteration of intestinal epithelial tight junction protein expression. The question arises as to whether PEs can cause irreversible epithelial damage and tight junction openings sufficient to permit co-absorption of payloads with bystander pathogens, lipopolysaccharides and its fragment, or exo- and endotoxins that may be associated with sepsis, inflammation and autoimmune conditions. Most PEs seem to cause membrane perturbation to varying extents that is rapidly reversible, and overall evidence of pathogen co-absorption is generally lacking. It is unknown however, whether the intestinal epithelial damage-repair cycle is sustained during repeat-dosing regimens for chronic therapy.

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

PubMed

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

2013-12-01

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

Activation of G protein-coupled estrogen receptor 1 (GPER-1) ameliorates blood-brain barrier permeability after global cerebral ischemia in ovariectomized rats.

PubMed

Lu, Dan; Qu, Yan; Shi, Fei; Feng, Dayun; Tao, Kai; Gao, Guodong; He, Shiming; Zhao, Tianzhi

2016-08-19

G protein-coupled estrogen receptor 1 (GPER-1) plays important roles in estrogen-mediated neuroprotection. However, protective effects of GPER-1 on blood-brain barrier (BBB) after ischemic stroke have not been determined. The aim of present study was to determine whether GPER-1 activation ameliorates BBB permeability in ovariectomized rats with induced global cerebral ischemia (GCI). GCI was induced by 4-vessel occlusion for 20 min followed by 24 h reperfusion period. The GPER-1 agonist (G1) was bilaterally administered immediately upon reperfusion by intracerebroventricular (icv) injection. We found that the GPER-1 agonist could significantly decrease immunoglobulin G (IgG) extravasation and increase the levels of tight junctions (occludin and claudin-5) in the CA1 at 24 h of reperfusion after GCI. Further, protein levels of vascular endothelial growth factor A (VEGF-A) was significantly decreased in the ischemic CA1 by G1. Our results suggest that GPER-1 activation reduce tight junctions disruption via inhibition of VEGF-A expression after ischemic injury. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2018-01-01

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

Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity.

PubMed

Palus, Martin; Vancova, Marie; Sirmarova, Jana; Elsterova, Jana; Perner, Jan; Ruzek, Daniel

2017-07-01

Alteration of the blood-brain barrier (BBB) is a hallmark of tick-borne encephalitis (TBE), a life-threating human viral neuroinfection. However, the mechanism of BBB breakdown during TBE, as well as TBE virus (TBEV) entry into the brain is unclear. Here, primary human microvascular endothelial cells (HBMECs) were infected with TBEV to study interactions with the BBB. Although the number of infected cells was relatively low in culture (<5%), the infection was persistent with high TBEV yields (>10 6 pfu/ml). Infection did not induce any significant changes in the expression of key tight junction proteins or upregulate the expression of cell adhesion molecules, and did not alter the highly organized intercellular junctions between HBMECs. In an in vitro BBB model, the virus crossed the BBB via a transcellular pathway without compromising the integrity of the cell monolayer. The results indicate that HBMECs may support TBEV entry into the brain without altering BBB integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

Higgins, Adam Z.; Karlsson, Jens O.M.

2013-01-01

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

Virus interaction with the apical junctional complex.

PubMed

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

2009-01-01

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

Expression of drebrin, an actin binding protein, in basal cell carcinoma, trichoblastoma and trichoepithelioma.

PubMed

Mizutani, Yoko; Iwamoto, Ikuko; Kanoh, Hiroyuki; Seishima, Mariko; Nagata, Koh-ichi

2014-06-01

Drebrin, an F-actin binding protein, is known to play important roles in cell migration, synaptogenesis and neural plasticity. Although drebrin was long thought to be specific for neuronal cells, its expression has recently been reported in non-neuronal cells. As for skin-derived cells, drebrin was shown to be enriched at adhering junctions (AJs) in cultured primary keratinocytes and also be highly expressed in basal cell carcinoma (BCC) cells. Since BCC and two types of benign neoplasm, trichoblastoma and trichoepithelioma, are considered to derive from the same origin, follicular germinative cells, it is sometimes difficult to morphologically distinguish BCC from trichoblastoma and trichoepithelioma. In this study, we performed immunohistochemical staining of drebrin in BCC, trichoblastoma and trichoepithelioma, to examine whether drebrin could serve as a biomarker for BCC diagnosis. In western blotting, drebrin was detected highly and moderately in the lysates from a squamous cell carcinoma cell line, DJM-1, and normal human epidermis, respectively. In immunofluorescence analyses, drebrin was colocalized with markers of AJs and tight junctions in DJM-1 cells and detected at cell-cell junction areas of human normal epidermis tissue. We then examined the distribution patterns of drebrin in BCC, trichoblastoma and trichoepithelioma. In BCC tissues, intense and homogeneous drebrin expression was observed mainly at tumor cell-cell boundaries. In contrast, drebrin was stained only weakly and non-homogeneously in trichoblastoma and trichoepthelioma tissue samples. For differential diagnosis of BCC, drebrin may be a novel and useful marker.

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

PubMed

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

2018-04-14

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

Tight junction-based epithelial microenvironment and cell proliferation.

PubMed

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

2008-11-24

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

ProbeZT: Simulation of transport coefficients of molecular electronic junctions under environmental effects using Büttiker's probes

NASA Astrophysics Data System (ADS)

Korol, Roman; Kilgour, Michael; Segal, Dvira

2018-03-01

We present our in-house quantum transport package, ProbeZT. This program provides linear response coefficients: electrical and electronic thermal conductances, as well as the thermopower of molecular junctions in which electrons interact with the surrounding thermal environment. Calculations are performed based on the Büttiker probe method, which introduces decoherence, energy exchange and dissipation effects phenomenologically using virtual electrode terminals called probes. The program can realize different types of probes, each introducing various environmental effects, including elastic and inelastic scattering of electrons. The molecular system is described by an arbitrary tight-binding Hamiltonian, allowing the study of different geometries beyond simple one-dimensional wires. Applications of the program to study the thermoelectric performance of molecular junctions are illustrated. The program also has a built-in functionality to simulate electron transport in double-stranded DNA molecules based on a tight-binding (ladder) description of the junction.

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

PubMed Central

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

2013-01-01

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

Fermented Herbal Formulas KIOM-MA128 Ameliorate IL-6-Induced Intestinal Barrier Dysfunction in Colon Cancer Cell Line

PubMed Central

Park, Kwang Il; Kim, Dong Gun; Lee, Bo Hyoung

2016-01-01

Inflammatory bowel disease (IBD) comprises Crohn's disease (CD) and ulcerative colitis (UC). IBD increases the risk of colorectal cancer (CRC), depending on the extent and duration of intestinal inflammation. Increased IL-6 expression has been reported in IBD patients, which may be associated with intestinal barrier function through discontinuous tight junction (TJ). KIOM-MA is a specific agent for allergic diseases and cancer, and it is composed of several plants; these herbs have been used in traditional oriental medicine. We fermented KIOM-MA, the product of KIOM-MA128, using probiotics to improve the therapeutic efficacy via the absorption and bioavailability of the active ingredients. In this study, we demonstrated that KIOM-MA/MA128 exhibited anticolitis effects via the modulation of TJ protein. Interleukin-6 resulted in a dose-dependent decrease in the TER and an increase in the FITC-dextran permeability; however, pretreatment with 400 µg/ml KIOM-MA/MA128 resulted in a significant increase in the TER and a decrease in the FITC-dextran permeability via IL-6 induction. Furthermore, protein and mRNA TJ levels remained stable after pretreatment with 400 µg/ml KIOM-MA/MA128. Moreover, KIOM-MA/MA128 suppressed the expression of PLCγ1 and PKC. Taken together, these findings suggest novel information and clue of the anticolitis effects of KIOM-MA128 via regulation of tight junction. PMID:27980357

Fermented Herbal Formulas KIOM-MA128 Ameliorate IL-6-Induced Intestinal Barrier Dysfunction in Colon Cancer Cell Line.

PubMed

Park, Kwang Il; Kim, Dong Gun; Lee, Bo Hyoung; Ma, Jin Yeul

2016-01-01

Inflammatory bowel disease (IBD) comprises Crohn's disease (CD) and ulcerative colitis (UC). IBD increases the risk of colorectal cancer (CRC), depending on the extent and duration of intestinal inflammation. Increased IL-6 expression has been reported in IBD patients, which may be associated with intestinal barrier function through discontinuous tight junction (TJ). KIOM-MA is a specific agent for allergic diseases and cancer, and it is composed of several plants; these herbs have been used in traditional oriental medicine. We fermented KIOM-MA, the product of KIOM-MA128, using probiotics to improve the therapeutic efficacy via the absorption and bioavailability of the active ingredients. In this study, we demonstrated that KIOM-MA/MA128 exhibited anticolitis effects via the modulation of TJ protein. Interleukin-6 resulted in a dose-dependent decrease in the TER and an increase in the FITC-dextran permeability; however, pretreatment with 400  µ g/ml KIOM-MA/MA128 resulted in a significant increase in the TER and a decrease in the FITC-dextran permeability via IL-6 induction. Furthermore, protein and mRNA TJ levels remained stable after pretreatment with 400  µ g/ml KIOM-MA/MA128. Moreover, KIOM-MA/MA128 suppressed the expression of PLC γ 1 and PKC. Taken together, these findings suggest novel information and clue of the anticolitis effects of KIOM-MA128 via regulation of tight junction.

Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum.

PubMed

Li, Hongxin; Liu, Xiaolin; Chen, Feiyang; Zuo, Kejing; Wu, Che; Yan, Yiming; Chen, Weiguo; Lin, Wencheng; Xie, Qingmei

2018-05-18

Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli . This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia , especially E. coli , significantly increased ( p < 0.01) at five days post-infection (dpi), while Lactobacillus , Enterococcus , and other probiotic organisms were significantly reduced ( p < 0.01). Simultaneously, the mRNA expression of tight junction proteins ( ZO-1 , claudin 3, and occludin), TFF2, and Muc2 were significantly reduced ( p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection.

Effect of tributyltin on mammalian endothelial cell integrity.

PubMed

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

2015-01-01

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

Shifts in Host Mucosal Innate Immune Function Are Associated with Ruminal Microbial Succession in Supplemental Feeding and Grazing Goats at Different Ages

PubMed Central

Jiao, Jinzhen; Zhou, Chuanshe; Guan, L. L.; McSweeney, C. S.; Tang, Shaoxun; Wang, Min; Tan, Zhiliang

2017-01-01

Gastrointestinal microbiota may play an important role in regulating host mucosal innate immune function. This study was conducted to test the hypothesis that age (non-rumination, transition and rumination) and feeding type [Supplemental feeding (S) vs. Grazing (G)] could alter ruminal microbial diversity and maturation of host mucosal innate immune system in goat kids. MiSeq sequencing was applied to investigate ruminal microbial composition and diversity, and RT-PCR was used to test expression of immune-related genes in ruminal mucosa. Results showed that higher (P < 0.05) relative abundances of Prevotella, Butyrivibrio, Pseudobutyrivibrio, Methanobrevibacter.gottschalkii, Neocallimastix, Anoplodinium–Diplodinium, and Polyplastron, and lower relative abundance of Methanosphaera (P = 0.042) were detected in the rumen of S kids when compared to those in G kids. The expression of genes encoding TLRs, IL1α, IL1β and TICAM2 was down-regulated (P < 0.01), while expression of genes encoding tight junction proteins was up-regulated (P < 0.05) in the ruminal mucosa of S kids when compared to that in G kids. Moreover, irrespective of feeding type, relative abundances of ruminal Prevotella, Fibrobacter, Ruminococcus, Butyrivibrio, Methanobrevibacter, Neocallimastix, and Entodinium increased with age. The expression of most genes encoding TLRs and cytokines increased (P < 0.05) from day 0 to 7, while expression of genes encoding tight junction proteins declined with age (P < 0.05). This study revealed that the composition of each microbial domain changed as animals grew, and these changes might be associated with variations in host mucosal innate immune function. Moreover, supplementing goat kids with concentrate could modulate ruminal microbial composition, enhance barrier function and decrease local inflammation. The findings provide useful information in interpreting microbiota and host interactions, and developing nutritional strategies to improve the productivity and health of rumen during early life. PMID:28912767

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

PubMed

Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

2015-04-01

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Glutamine supplementation improves intestinal barrier function in a weaned piglet model of Escherichia coli infection.

PubMed

Ewaschuk, Julia B; Murdoch, Gordon K; Johnson, Ian R; Madsen, Karen L; Field, Catherine J

2011-09-01

The weaning period is associated with an increased prevalence of gastrointestinal infection in many species. Glutamine (Gln) has been shown to improve intestinal barrier function and immune function in both in vivo and in vitro models. The objective of the present study was to determine the effect of dietary Gln supplementation on intestinal barrier function and intestinal cytokines in a model of Escherichia coli infection. We randomised 21-d-old piglets (n 20) to nutritionally complete isonitrogenous diets with or without Gln (4·4 %, w/w) for 2 weeks. Intestinal loops were isolated from anaesthetised pigs and inoculated with either saline or one of the two E. coli (K88AC or K88 wild-type)-containing solutions. Intestinal tissue was studied for permeability, cytokine expression, fluid secretion and tight-junction protein expression. Animals receiving Gln supplementation had decreased potential difference (PD) and short-circuit current (I(sc)) in E. coli-inoculated intestinal loops (PD 0·628 (SEM 0·151) mV; I(sc) 13·0 (SEM 3·07) μA/cm(2)) compared with control-fed animals (PD 1·36 (SEM 0·227) mV; I(sc) 22·4 (SEM 2·24) μA/cm(2)). Intestinal tissue from control, but not from Gln-supplemented, animals responded to E. coli with a significant increase in mucosal cytokine mRNA (IL-1β, IL-6, transforming growth factor-β and IL-10). Tight-junction protein expression (claudin-1 and occludin) was reduced with exposure to E. coli in control-fed animals and was not influenced in Gln-supplemented piglets. Gln supplementation may be useful in reducing the severity of weaning-related gastrointestinal infections, by reducing the mucosal cytokine response and altering intestinal barrier function.

Lead exposure results in hearing loss and disruption of the cochlear blood-labyrinth barrier and the protective role of iron supplement.

PubMed

Liu, Xinqin; Zheng, Gang; Wu, Yongxiang; Shen, Xuefeng; Jing, Jinfei; Yu, Tao; Song, Han; Chen, Jingyuan; Luo, Wenjing

2013-12-01

This study was designed to investigate the impact of lead (Pb(2+)) on the auditory system and its molecular mechanisms. Pb(AC)2 was administrated to male SD rats aged 21-22 d for 8 weeks at a dose of 300ppm. Male guinea pigs were also administrated with 50mg/kg Pb(AC)2 two times a week for 8 weeks. The auditory nerve-brainstem evoked responses (ABR) was recorded and the morphological changes of the outer hair cells (OHCs) were observed with Phallodin-FITC staining. In addition, the integrity of the blood-labyrinth barrier was observed by TEM and the expression of tight junction proteins (TJPs) in the cochlear stria vascularis was determined by immunofluorescence. Our results showed that Pb(2+) exposure resulted in increased ABR threshold in both rats and guinea pigs. Abnormal shapes and loss of OHCs were found in the cochlear basilar membrane following the Pb(2+) exposure. TEM study showed that the tight junctions between the endothelial cells and the border cells were lost and disrupted. Down-regulation of the occludin, ZO-1 and claudin-5 in the stria vascularis suggested that the increased permeability of the blood-labyrinth barrier may attribute to the Pb(2+)-induced decrease of TJPs' expression. Additionally, Fe(2+) supplement partly reversed the Pb(2+)-induced hearing loss and down-regulation of TJPs. Taken together, these data indicate that the disruption of blood-labyrinth barrier by down-regulating the expression of TJPs plays a role in the Pb(2+)-induced hearing loss, and Fe(2+) supplement protects the auditory system against Pb(2+)-induced toxicity and may have significant clinical implications. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed

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

2010-01-01

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

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

PubMed

Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

2016-06-01

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

Beneficial effects of Rifaximin in post-infectious irritable bowel syndrome mouse model beyond gut microbiota.

PubMed

Jin, Yu; Ren, Xiaoyang; Li, Gangping; Li, Ying; Zhang, Lei; Wang, Huan; Qian, Wei; Hou, Xiaohua

2018-02-01

Rifaximin is a minimally absorbed antibiotic, which has shown efficacy in irritable bowel syndrome (IBS) patients. However, the mechanism on how it effects in IBS is still incompletely defined. In this study, Trichinella spiralis-infected post-infectious (PI) IBS mouse model was used, to assess the action of rifaximin on visceral hypersensitivity, barrier function, gut inflammation, and microbiota. Post-infectious IBS model was established by T. spiralis infection in mice. Rifaximin were administered to PI-IBS mice for seven consecutive days. The abdominal withdrawal reflex and threshold of colorectal distention were employed to evaluate visceral sensitivity. Smooth muscle contractile response was recorded in the organ bath. Intestinal permeability was measured by Ussing chamber. Expression of tight junction protein and cytokines were measured by Western blotting. Ilumina miseq platform was used to analyze bacterial 16S ribosomal RNA. Post-infectious IBS mice treated with rifaximin exhibited decreased abdominal withdrawal reflex score, increased threshold, reduced contractile response, and intestinal permeability. Rifaximin also suppressed the expression of interleukin-12 and interleukin-17 and promoted the expression of the major tight junction protein occludin. Furthermore, rifaximin did not change the composition and diversity, and the study reavealed that rifaximin had a tiny effect on the relative abundance of Lactobacillus and Bifidobacterium in this PI-IBS model. Rifaximin alleviated visceral hypersensitivity, recovered intestinal barrier function, and inhibited low-grade inflammation in colon and ileum of PI-IBS mouse model. Moreover, rifaximin exerts anti-inflammatory effects with only a minimal effect on the overall composition and diversity of the gut microbiota in this model. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

PubMed

Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

2016-01-01

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

Matrix Metalloproteinase-Mediated Blood-Brain Barrier Dysfunction in Epilepsy.

PubMed

Rempe, Ralf G; Hartz, Anika M S; Soldner, Emma L B; Sokola, Brent S; Alluri, Satya R; Abner, Erin L; Kryscio, Richard J; Pekcec, Anton; Schlichtiger, Juli; Bauer, Björn

2018-05-02

The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo / ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A 2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage. SIGNIFICANCE STATEMENT The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A 2 , which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden. Copyright © 2018 the authors 0270-6474/18/384301-15$15.00/0.

The effects of electromagnetic pulse on the protein levels of tight junction associated-proteins in the cerebral cortex, hippocampus, heart, lung, and testis of rats.

PubMed

Qiu, LianBo; Chen, Chen; Ding, GuiRong; Zhou, Yan; Zhang, MengYao

2011-08-01

To investigate changes in the expression of tight junction (TJ) proteins in the cerebral cortex, hippocampus, heart, lung, and testes of rats after exposure to electromagnetic pulse (EMP). Eighteen adult male Sprague-Dawley rats were divided into sham and exposure groups. The exposure groups received EMP at 200 kV/m for 200 pulses with a repetition rate of 1 Hz. The expression of TJ proteins (ZO-1, occludin, actin) in the several organs was examined by western blotting. ZO-1 levels in the cerebral cortex decreased 1 h and 3 h after EMP exposure compared with sham group (P<0.05). No significant difference was observed for occludin and actin. ZO-1 levels in the hippocampus increased 1 h and 3 h post-exposure (P<0.05), and occludin decreased after 3 h (P<0.05); however, actin was unaffected. ZO-1 levels in the heart increased 3 h post-exposure (P<0.05), occludin decreased 3 h post-exposure (P<0.05), and actin increased 1 h and 3 h post-exposure (P<0.05). ZO-1, occludin and actin levels in the lung decreased compared with those in the sham group (P<0.05). ZO-1 and occludin levels in the testes decreased 1 h and 3 h post-exposure (P<0.05), but actin showed no significant change. Exposure to EMP altered the expression levels of TJ proteins, particularly ZO-1, in the organs of adult male rats, which may induce changes in barrier structure and function. Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

PubMed

Perez White, Bethany E; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J; Thomas, Paul M; Getsios, Spiro

2017-01-01

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. © 2017. Published by The Company of Biologists Ltd.

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

PubMed

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

2016-02-01

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

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions

PubMed Central

Perez White, Bethany E.; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J.; Thomas, Paul M.

2017-01-01

ABSTRACT EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. PMID:27815408

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

PubMed Central

2013-01-01

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

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

PubMed

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

2013-11-22

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

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

PubMed Central

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

2005-01-01

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

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

PubMed

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

2005-09-01

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

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

PubMed

Ohtsuki, Sumio; Ikeda, Chiemi; Uchida, Yasuo; Sakamoto, Yumi; Miller, Florence; Glacial, Fabienne; Decleves, Xavier; Scherrmann, Jean-Michel; Couraud, Pierre-Olivier; Kubo, Yoshiyuki; Tachikawa, Masanori; Terasaki, Tetsuya

2013-01-07

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Interfacial spin-filter assisted spin transfer torque effect in Co/BeO/Co magnetic tunnel junction

NASA Astrophysics Data System (ADS)

Tang, Y.-H.; Chu, F.-C.

2015-03-01

The first-principles calculation is employed to demonstrate the spin-selective transport properties and the non-collinear spin-transfer torque (STT) effect in the newly proposed Co/BeO/Co magnetic tunnel junction. The subtle spin-polarized charge transfer solely at O/Co interface gives rise to the interfacial spin-filter (ISF) effect, which can be simulated within the tight binding model to verify the general expression of STT. This allows us to predict the asymmetric bias behavior of non-collinear STT directly via the interplay between the first-principles calculated spin current densities in collinear magnetic configurations. We believe that the ISF effect, introduced by the combination between wurtzite-BeO barrier and the fcc-Co electrode, may open a new and promising route in semiconductor-based spintronics applications.

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

Crawford, Melissa; Dagnino, Lina

2017-10-02

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

Porcine circovirus 2 (PCV2) increases the expression of endothelial adhesion/junction molecules.

PubMed

Marks, Fernanda S; Almeida, Laura L; Driemeier, David; Canal, Cláudio; Barcellos, David E S N; Guimarães, Jorge A; Reck, José

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus disease, a complex multisystem syndrome in domestic pigs. Despite the significant economic losses caused by porcine circovirus disease, the mechanisms of pathogenesis underlying the clinical findings remain largely unclear. As various reports have highlighted the potential key role of vascular lesions in the pathogenesis of porcine circovirus disease, the aim of this work was to investigate effects of PCV2 infection on vascular endothelial cells, focusing on cell viability and expression of adhesion/junction molecules. PCV2 infection reduced endothelial cell viability, while viral infection did not affected the viability of several other classical cell lines. Also, PCV2 infection in endothelial cells displayed a dual/biphasic effect: initially, infection increased ICAM-1 expression, which can favor leukocyte recruitment and emigration to tissues and possibly inducing characteristic porcine circovirus disease inflammatory lesions; then, secondarily, infection caused an increase in zonula occludens 1 tight junction protein (ZO-1) expression, which in turn can result in difficulties for cell traffic across the endothelium and a potential impairment the immune response in peripheral tissues. These virus-induced endothelial changes could directly impact the inflammatory process of porcine circovirus disease and associated vascular/immune system disturbances. Data suggest that, among the wide range of effects induced by PCV2 on the host, endothelial modulation can be a pivotal process which can help to explain PCV2 pathogenesis in some porcine circovirus disease presentations. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines.

PubMed

Baek, Hyekyung; Kim, Kwang Ho; Park, Min Young; Kim, Kyeongryun; Ko, Bokyeong; Seo, Hyung Seok; Kim, Byoung Soo; Hahn, Tae-Wook; Yi, Sun Shin

2017-08-31

With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS ( Salmonella and Escherichia ). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines.

Formononetin Administration Ameliorates Dextran Sulfate Sodium-Induced Acute Colitis by Inhibiting NLRP3 Inflammasome Signaling Pathway.

PubMed

Wu, Dacheng; Wu, Keyan; Zhu, Qingtian; Xiao, Weiming; Shan, Qing; Yan, Zhigang; Wu, Jian; Deng, Bin; Xue, Yan; Gong, Weijuan; Lu, Guotao; Ding, Yanbing

2018-01-01

Formononetin is a kind of isoflavone compound and has been reported to possess anti-inflammatory properties. In this present study, we aimed to explore the protective effects of formononetin on dextran sulfate sodium- (DSS-) induced acute colitis. By intraperitoneal injection of formononetin in mice, the disease severity of colitis was attenuated in a dose-dependent manner, mainly manifesting as relieved clinical symptoms of colitis, mitigated colonic epithelial cell injury, and upregulations of colonic tight junction proteins levels (ZO-1, claudin-1, and occludin). Meanwhile, our study found that formononetin significantly prevented acute injury of colonic cells induced by TNF- α in vitro, specifically manifesting as the increased expressions of colonic tight junction proteins (ZO-1, claudin-1, and occludin). In addition, the result showed that formononetin could reduce the NLRP3 pathway protein levels (NLRP3, ASC, IL-1 β ) in vivo and vitro, and MCC950, the NLRP3 specific inhibitor, could alleviate the DSS-induced mice acute colitis. Furthermore, in the foundation of administrating MCC950 to inhibit activation of NLRP3 inflammasome, we failed to observe the protective effects of formononetin on acute colitis in mice. Collectively, our study for the first time confirmed the protective effects of formononetin on DSS-induced acute colitis via inhibiting the NLRP3 inflammasome pathway activation.

Lentiviral transduction of rat Sertoli cells as a means to modify gene expression

PubMed Central

Nicholls, Peter K.; Stanton, Peter G.; Rainczuk, Katarzyna E.; Qian, Hongwei; Gregorevic, Paul; Harrison, Craig A.

2012-01-01

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications. PMID:23248769

Claudins, dietary milk proteins, and intestinal barrier regulation.

PubMed

Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

2013-01-01

The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. © 2013 International Life Sciences Institute.

Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells.

PubMed

Radwanska, Agata; Grall, Dominique; Schaub, Sébastien; Divonne, Stéphanie Beghelli-de la Forest; Ciais, Delphine; Rekima, Samah; Rupp, Tristan; Sudaka, Anne; Orend, Gertraud; Van Obberghen-Schilling, Ellen

2017-10-06

Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells.

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

PubMed Central

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

2017-01-01

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

Functional analysis of tight junction organization.

PubMed

DiBona, D R

1985-01-01

The functional basis of tight junction design has been examined from the point of view that this rate-limiting barrier to paracellular transport is a multicompartment system. Review of the osmotic sensitivity of these structures points to the need for this sort of analysis for meaningful correlation of structure and function under a range of conditions. A similar conclusion is drawn with respect to results from voltage-clamping protocols where reversal of spontaneous transmural potential difference elicits parallel changes in both structure and function in much the same way as does reversal of naturally occurring osmotic gradients. In each case, it becomes necessary to regard the junction as a functionally polarized structure to account for observations of its rectifying properties. Lastly, the details of experimentally-induced junction deformation are examined in light of current theories of its organization; arguments are presented in favor of the view that the primary components of intramembranous organization (as viewed with freeze-fracture techniques) are lipidic rather than proteinaceous.

[Study of the effect of surface-active agents on living cells, used as component parts in microemulsions, based on their chemical structures and critical micelle-formative concentration (CMC)].

PubMed

Ujhelyi, Zoltán; Vecsernyés, Miklós; Bácskay, Ildikó

2013-01-01

The aim of this study was to examine the cellular effects of two nonionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group:Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase the solubility of hydrophobic drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Caco-2 cell monolayers are convenient and reliable in vitro models of the gastrointestinal tract. Paracellular permeability was examined with Lucifer yellow assays. The integrity of cell monolayers was observed by TransEpithelial Electrical Resistance (TEER) measurements. Tight junction alterations effected by the surfactants were also characterized as evidence for paracellular pathway. Changes in sub cellular localization of the tight junction proteins: ZO-1, Claudin-land beta-cathenin, were examined by confocal laser scanning microscopy.The results of cytotoxicity assays were in agreement and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol, the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol--0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC50 concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

Paracellular transport in the collecting duct

PubMed Central

Hou, Jianghui

2016-01-01

Purpose of review The paracellular pathway through the tight junction provides an important route for chloride reabsorption in the collecting duct of the kidney. This review describes recent findings of how defects in paracellular chloride permeation pathway may cause kidney diseases and how such a pathway may be regulated to maintain normal chloride homeostasis. Recent findings The tight junction in the collecting duct expresses two important claudin genes – claudin-4 and claudin-8. Transgenic knockout of either claudin gene causes hypotension, hypochloremia, and metabolic alkalosis in experimental animals. The claudin-4 mediated chloride permeability can be regulated by a protease endogenously expressed by the collecting duct cell – Cap1. Cap1 regulates the intercellular interaction of claudin-4 and its membrane stability. KLHL3, previously identified as a causal gene for Gordon’s syndrome, also known as pseudohypoaldosteronism II (PHA-II), directly interacts with claudin-8 and regulates its ubiquitination and degradation. The dominant PHA-II mutation (R528H) in KLHL3 abolishes claudin-8 binding, ubiquitination, and degradation. Summary The paracellular chloride permeation pathway in the kidney is an important but understudied area in nephrology. It plays vital roles in renal salt handling and regulation of extracellular fluid volume and blood pressure. Two claudin proteins – claudin-4 and claudin-8 contribute to the function of this paracellular pathway. Deletion of either claudin protein from the collecting duct causes renal chloride reabsorption defects and low blood pressure. Claudins can be regulated on post-translational levels by several mechanisms involving protease and ubiquitin ligase. Deregulation of claudins may cause human hypertension as exemplified in the Gordon’s syndrome. PMID:27490784

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

PubMed

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

2014-01-01

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

Effect of lactobacilli on paracellular permeability in the gut.

PubMed

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

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

Edge currents in frustrated Josephson junction ladders

NASA Astrophysics Data System (ADS)

Marques, A. M.; Santos, F. D. R.; Dias, R. G.

2016-09-01

We present a numerical study of quasi-1D frustrated Josephson junction ladders with diagonal couplings and open boundary conditions, in the large capacitance limit. We derive a correspondence between the energy of this Josephson junction ladder and the expectation value of the Hamiltonian of an analogous tight-binding model, and show how the overall superconducting state of the chain is equivalent to the minimum energy state of the tight-binding model in the subspace of one-particle states with uniform density. To satisfy the constraint of uniform density, the superconducting state of the ladder is written as a linear combination of the allowed k-states of the tight-binding model with open boundaries. Above a critical value of the parameter t (ratio between the intra-rung and inter-rung Josephson couplings) the ladder spontaneously develops currents at the edges, which spread to the bulk as t is increased until complete coverage is reached. Above a certain value of t, which varies with ladder size (t = 1 for an infinite-sized ladder), the edge currents are destroyed. The value t = 1 corresponds, in the tight-binding model, to the opening of a gap between two bands. We argue that the disappearance of the edge currents with this gap opening is not coincidental, and that this points to a topological origin for these edge current states.

Myosin light chain kinase knockout improves gut barrier function and confers a survival advantage in polymicrobial sepsis.

PubMed

Lorentz, C Adam; Liang, Zhe; Meng, Mei; Chen, Ching-Wen; Yoseph, Benyam P; Breed, Elise R; Mittal, Rohit; Klingensmith, Nathan J; Farris, Alton B; Burd, Eileen M; Koval, Michael; Ford, Mandy L; Coopersmith, Craig M

2017-06-07

Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK -/- and wild type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK -/- mice (95% vs. 24%, p<0.0001). Intestinal permeability increased in septic WT mice compared to unmanipulated mice. In contrast, permeability in septic MLCK -/- mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK -/- mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8, 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK -/- mice; however, survival was similar between septic MLCK -/- mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.

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

PubMed

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

2013-08-01

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

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

PubMed

Sha, Xian-Yi; Fang, Xiao-Ling

2006-01-01

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

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.

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

PubMed Central

Alberini, Giulio; Benfenati, Fabio

2017-01-01

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

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

PubMed

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

2016-04-19

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

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.

Effects of Osmolality on Paracellular Transport in MDCK II Cells

PubMed Central

Hirai, Toyohiro; Furuse, Mikio

2016-01-01

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

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

PubMed Central

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

2011-01-01

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

Mechanisms of the Testis Toxicity Induced by Chronic Exposure to Mequindox.

PubMed

Liu, Qianying; Lei, Zhixin; Huang, Anxiong; Lu, Qirong; Wang, Xu; Ahmed, Saeed; Awais, Ihsan; Yuan, Zonghui

2017-01-01

Mequindox (MEQ) is a synthetic antimicrobial agent widely used in China since the 1980s. Although the toxicity of MEQ is well recognized, its testis toxicity has not been adequately investigated. In the present study, we provide evidence that MEQ triggers oxidative stress, mitochondrion dysfunction and spermatogenesis deficiency in mice after exposure to MEQ (0, 25, 55, and 110 mg/kg in the diet) for up to 18 months. The genotoxicity and adrenal toxicity may contribute to sperm abnormalities caused by MEQ. Moreover, using LC/MS-IT-TOF analysis, two metabolites, 3-methyl-2-(1-hydroxyethyl) quinoxaline- N 4-monoxide (M4) and 3-methyl-2-(1-hydroxyethyl) quinoxaline- N 1-monoxide (M8), were detected in the serum of mice, which directly confirms the relationship between the N →O group reduction metabolism of MEQ and oxidative stress. Interestingly, only M4 was detected in the testes, suggesting that the higher reproductive toxicity of M4 than M8 might be due to the increased stability of M4-radical (M4-R) compared to M8-radical (M8-R). Furthermore, the expression of the blood-testis barrier (BTB)-associated junctions such as tight junctions, gap junctions and basal ectoplasmic specializations were also examined. The present study demonstrated for the first time the role of the M4 in testis toxicity, and illustrated that the oxidative stress, mitochondrion dysfunction and interference in spermatogenesis, as well as the altered expression of BTB related junctions, were involved in the reproductive toxicity mediated by MEQ in vivo .

Mechanisms of the Testis Toxicity Induced by Chronic Exposure to Mequindox

PubMed Central

Liu, Qianying; Lei, Zhixin; Huang, Anxiong; Lu, Qirong; Wang, Xu; Ahmed, Saeed; Awais, Ihsan; Yuan, Zonghui

2017-01-01

Mequindox (MEQ) is a synthetic antimicrobial agent widely used in China since the 1980s. Although the toxicity of MEQ is well recognized, its testis toxicity has not been adequately investigated. In the present study, we provide evidence that MEQ triggers oxidative stress, mitochondrion dysfunction and spermatogenesis deficiency in mice after exposure to MEQ (0, 25, 55, and 110 mg/kg in the diet) for up to 18 months. The genotoxicity and adrenal toxicity may contribute to sperm abnormalities caused by MEQ. Moreover, using LC/MS-IT-TOF analysis, two metabolites, 3-methyl-2-(1-hydroxyethyl) quinoxaline-N4-monoxide (M4) and 3-methyl-2-(1-hydroxyethyl) quinoxaline-N1-monoxide (M8), were detected in the serum of mice, which directly confirms the relationship between the N→O group reduction metabolism of MEQ and oxidative stress. Interestingly, only M4 was detected in the testes, suggesting that the higher reproductive toxicity of M4 than M8 might be due to the increased stability of M4-radical (M4-R) compared to M8-radical (M8-R). Furthermore, the expression of the blood-testis barrier (BTB)-associated junctions such as tight junctions, gap junctions and basal ectoplasmic specializations were also examined. The present study demonstrated for the first time the role of the M4 in testis toxicity, and illustrated that the oxidative stress, mitochondrion dysfunction and interference in spermatogenesis, as well as the altered expression of BTB related junctions, were involved in the reproductive toxicity mediated by MEQ in vivo. PMID:29018347

Myosin-X functions in polarized epithelial cells

PubMed Central

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

2012-01-01

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

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

NASA Astrophysics Data System (ADS)

Bolinger, Mark Thomas

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

Effect of Lactobacilli on Paracellular Permeability in the Gut

PubMed Central

Ahrne, Siv; Hagslatt, Marie-Louise Johansson

2011-01-01

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

Delayed astrocytic contact with cerebral blood vessels in FGF-2 deficient mice does not compromise permeability properties at the developing blood-brain barrier.

PubMed

Saunders, Norman R; Dziegielewska, Katarzyna M; Unsicker, Klaus; Ek, C Joakim

2016-11-01

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016. © 2016 Wiley Periodicals, Inc.

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

PubMed

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

1999-04-01

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

Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function.

PubMed

Tsukita, Sachiko; Katsuno, Tatsuya; Yamazaki, Yuji; Umeda, Kazuaki; Tamura, Atsushi; Tsukita, Shoichiro

2009-05-01

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutes and ions through the paracellular pathway. The molecular architecture of TJs has gradually been unraveled in recent years, providing the basis for "barriology" (defined by Shoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins are now considered to be the essential basic components of TJ strands, with which other integral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated. Peripherally associated scaffolding proteins are required for the organization of the integral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great deal of attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reported to be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadin and alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in mice and cells, which have provided clear and important information regarding the functions of ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression, simultaneous suppression of all three proteins has revealed the essential and nonessential in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specific TJ localization in a ZO-1/2-dependent fashion. ZO-1 and ZO-2 play pivotal roles in the final establishment of the belt-like adherens junctions (zonula adherens), followed by the formation of the belt-like TJs (zonula occludens) with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells.

Effect of retinoic acid on the tight junctions of the retinal pigment epithelium-choroid complex of guinea pigs with lens-induced myopia in vivo

PubMed Central

WANG, SHA; LIU, SHUANGZHEN; MAO, JUNFENG; WEN, DAN

2014-01-01

Zonula occludens-1 (ZO-1) and occludin are important tight junction (TJ)-associated proteins, which are expressed in the retinal pigment epithelium (RPE)-choroid complex. Retinoic acid (RA) is a regulator of eye growth and may play an important role in forming functional TJs. The aim of this study was to detect the changes that occur in the expression of ZO-1 and occludin in the RPE-choroid complex of guinea pigs with lens-induced myopia (LIM), and to investigate the effect of RA on TJ-associated proteins in vivo. We developed an animal model of myopia by placing a −6.00 D negative lens on the right eyes of 3-week-old guinea pigs. The refractive error and axial length of the eye were measured on days 0, 3, 7 and 14. High-performance liquid chromatography (HPLC) was performed to detect the changes in endogenous RA in the RPE-choroid complex. The expression of ZO-1 and occludin was observed by immunofluorescence and assayed by western blot analysis. Additionally, 2 μl LE540 (2.5 μg/μl), an antagonist of RA receptors (RARs), was injected into the vitreous chamber of the eyes of guinea pigs with LIM and 2 μl phosphate-buffered saline (PBS) (2.5 μg/μl) were injected as a negative control. We observed no obvious change in RA, ZO-1 and occludin expression in the normal control group within 14 days. In the LIM and LIM plus PBS groups, the level of RA and the expression of ZO-1 and occludin in the RPE-choroid complex significantly increased within 14 days along with the development of myopia. However, the level of RA was inhibited and the expression of TJ-associated proteins decreased in the eyes of guinea pigs with LIM following the injection of LE540. Thus, we consider that the expression of ZO-1 and occludin is increased in the RPE-choroid complex during the development of myopia. This change in expression may be regulated by RA, a factor known to be involved in the regulation of eye growth. PMID:24535401

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

PubMed

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

2013-10-09

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

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

PubMed

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

2014-09-01

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

Characterization of tight junction proteins in cultured human urothelial cells.

PubMed

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

2008-01-01

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

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

PubMed

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

2016-01-12

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

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

PubMed

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

2018-06-25

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

Prophylactic effect of rebamipide on aspirin-induced gastric lesions and disruption of tight junctional protein zonula occludens-1 distribution.

PubMed

Suzuki, Takahiro; Yoshida, Norimasa; Nakabe, Nami; Isozaki, Yutaka; Kajikawa, Hirokazu; Takagi, Tomohisa; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Naito, Yuji; Matsui, Hirofumi; Yoshikawa, Toshikazu

2008-03-01

Aspirin and nonsteroidal anti-inflammatory agents are known to induce gastroduodenal complications such as ulcer, bleeding, and dyspepsia. In this study, we examined the prophylactic effect of rebamipide, an anti-ulcer agent with free-radical scavenging and anti-inflammatory effect, on acidified aspirin-induced gastric mucosal injury in rats. In addition, we investigated the mucosal barrier functions disrupted by aspirin. Oral administration of acidified aspirin resulted in linear hemorrhagic erosions with increasing myeloperoxidase activity and thiobarbituric acid-reactive substance concentrations in the gastric mucosa. Rebamipide suppressed these acidified aspirin-induced gastric lesions and inflammatory changes significantly, and its protective effect was more potent in the case of repeated (twice daily for 3 days) treatment than single treatment before aspirin administration. Immunostaining of zonula occludens (ZO)-1, one of the tight junctional proteins, was strengthened in rat gastric mucosa after repeated administration of rebamipide. In addition, aspirin induced the increasing transport of fluorescine isothiocyanate-labeled dextrans with localized disruption and decreased expression of ZO-1 protein on rat gastric mucosal cell line RGM-1. Rebamipide effectively prevented aspirin-induced permeability changes and disruption of ZO-1 distribution. These results suggest that rebamipide protects against aspirin-induced gastric mucosal lesions by preserving gastric epithelial cell-to cell integrity in addition to the anti-inflammatory effects.

Role of claudins in renal calcium handling.

PubMed

Negri, Armando Luis

2015-01-01

Paracellular channels occurring in tight junctions play a major role in transepithelial ionic flows. This pathway includes a high number of proteins, such as claudins. Within renal epithelium, claudins result in an ionic selectivity in tight junctions. Ascending thick limb of loop of Henle (ATLH) is the most important segment for calcium reabsorption in renal tubules. Its cells create a water-proof barrier, actively transport sodium and chlorine through a transcellular pathway, and provide a paracellular pathway for selective calcium reabsorption. Several studies have led to a model of paracellular channel consisting of various claudins, particularly claudin-16 and 19. Claudin-16 mediates cationic paracellular permeability in ATLH, whereas claudin-19 increases cationic selectivity of claudin-16 by blocking anionic permeability. Recent studies have shown that claudin-14 promoting activity is only located in ATLH. When co-expressed with claudin-16, claudin-14 inhibits the permeability of claudin-16 and reduces paracellular permeability to calcium. Calcium reabsorption process in ATLH is closely regulated by calcium sensor receptor (CaSR), which monitors circulating Ca levels and adjusts renal excretion rate accordingly. Two microRNA, miR-9 and miR-374, are directly regulated by CaSR. Thus, miR-9 and miR-374 suppress mRNA translation for claudin-14 and induce claudin-14 decline. Copyright © 2015 The Author. Published by Elsevier España, S.L.U. All rights reserved.

Time-dependent expression of hypertonic effects on bullfrog taste nerve responses to salts and bitter substances.

PubMed

Mashiyama, Kazunori; Nozawa, Yuhei; Ohtubo, Yoshitaka; Kumazawa, Takashi; Yoshii, Kiyonori

2014-03-27

We previously showed that the hypertonicity of taste stimulating solutions modified tonic responses, the quasi-steady state component following the transient (phasic) component of each integrated taste nerve response. Here we show that the hypertonicity opens tight junctions surrounding taste receptor cells in a time-dependent manner and modifies whole taste nerve responses in bullfrogs. We increased the tonicity of stimulating solutions with non-taste substances such as urea or ethylene glycol. The hypertonicity enhanced phasic responses to NaCl>0.2M, and suppressed those to NaCl<0.1M, 1mM CaCl2, and 1mM bitter substances (quinine, denatonium and strychnine). The hypertonicity also enhanced the phasic responses to a variety of 0.5M salts such as LiCl and KCl. The enhancing effect was increased by increasing the difference between the ionic mobilities of the cations and anions in the salt. A preincubation time >20s in the presence of 1M non-taste substances was needed to elicit both the enhancing and suppressing effects. Lucifer Yellow CH, a paracellular marker dye, diffused into bullfrog taste receptor organs in 30s in the presence of hypertonicity. These results agreed with our proposed mechanism of hypertonic effects that considered the diffusion potential across open tight junctions. Copyright © 2014 Elsevier B.V. All rights reserved.

The angiopoietin1-Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8.

PubMed

Liu, Xinchun; Zhou, Xiaoshu; Yuan, Wei

2014-10-15

In mammalian central nervous system (CNS), the integrity of the blood-spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood-brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1-Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8. Copyright © 2014 Elsevier Inc. All rights reserved.

Formononetin Administration Ameliorates Dextran Sulfate Sodium-Induced Acute Colitis by Inhibiting NLRP3 Inflammasome Signaling Pathway

PubMed Central

Wu, Dacheng; Wu, Keyan; Zhu, Qingtian; Xiao, Weiming; Shan, Qing; Yan, Zhigang; Wu, Jian; Deng, Bin; Xue, Yan; Gong, Weijuan

2018-01-01

Formononetin is a kind of isoflavone compound and has been reported to possess anti-inflammatory properties. In this present study, we aimed to explore the protective effects of formononetin on dextran sulfate sodium- (DSS-) induced acute colitis. By intraperitoneal injection of formononetin in mice, the disease severity of colitis was attenuated in a dose-dependent manner, mainly manifesting as relieved clinical symptoms of colitis, mitigated colonic epithelial cell injury, and upregulations of colonic tight junction proteins levels (ZO-1, claudin-1, and occludin). Meanwhile, our study found that formononetin significantly prevented acute injury of colonic cells induced by TNF-α in vitro, specifically manifesting as the increased expressions of colonic tight junction proteins (ZO-1, claudin-1, and occludin). In addition, the result showed that formononetin could reduce the NLRP3 pathway protein levels (NLRP3, ASC, IL-1β) in vivo and vitro, and MCC950, the NLRP3 specific inhibitor, could alleviate the DSS-induced mice acute colitis. Furthermore, in the foundation of administrating MCC950 to inhibit activation of NLRP3 inflammasome, we failed to observe the protective effects of formononetin on acute colitis in mice. Collectively, our study for the first time confirmed the protective effects of formononetin on DSS-induced acute colitis via inhibiting the NLRP3 inflammasome pathway activation. PMID:29507526

Intraluminal polyethylene glycol stabilizes tight junctions and improves intestinal preservation in the rat.

PubMed

Oltean, M; Joshi, M; Björkman, E; Oltean, S; Casselbrant, A; Herlenius, G; Olausson, M

2012-08-01

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW-Viaspan. We investigated whether a low-sodium PEG solution is beneficial for intestines stored in histidine-tryptophane-ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG-3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush-border maltase activity, zonula occludens-1 (ZO-1) and claudin-3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush-border enzymes or promoting edema. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

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

PubMed Central

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

2013-01-01

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

Nrf2 activator ameliorates hemorrhagic transformation in focal cerebral ischemia under warfarin anticoagulation.

PubMed

Imai, Takahiko; Takagi, Toshinori; Kitashoji, Akira; Yamauchi, Keita; Shimazawa, Masamitsu; Hara, Hideaki

2016-05-01

Oxidative stress has been reported to be a main cause of neuronal cell death in ischemia reperfusion injury (IRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important factor involved in anti-oxidative responses. We previously reported that bardoxolone methyl (BARD), an Nrf2 activator, prevented damage induced by IRI. In this study, we investigated the effect of BARD on hemorrhagic transformation in the context of blood brain barrier (BBB) protection. Mice received pre-treatment with warfarin (4.0 mg/kg, p.o.). IRI was subsequently induced 18 h after the warfarin administration by transient middle cerebral artery occlusion (MCAO) for 6 h. BARD (0.06, 0.2, 0.6 or 2.0 mg/kg) or saline was injected intravenously immediately after reperfusion. The infarct volume, neurological score, intracranial hemorrhage volume, and BBB permeability were evaluated 24 h after MCAO. The survival rate and behavioral functional recovery were evaluated for 7 days following IRI. Furthermore, the effects of BARD on BBB components were investigated by western blotting and immunostaining analysis. BARD suppressed warfarin-mediated increases in the intracranial hemorrhage volume without affecting the infarct volume. BBB permeability was also suppressed by administration of BARD. Western blotting showed that BARD increased expression of BBB components such as endothelial cells, pericytes, and tight junction proteins. Furthermore, immunostaining showed that BARD induced localization of Nrf2 to endothelial cells and pericytes. BARD suppressed the exacerbation hemorrhage caused by warfarin pretreatment and ameliorated BBB disruption by protecting endothelial cells, pericytes, and tight junction protein expressions. These results indicate that Nrf2 activators may be an effective therapy against hemorrhagic transformation caused by anticoagulant drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Helminth infection in mice improves insulin sensitivity via modulation of gut microbiota and fatty acid metabolism.

PubMed

Pace, Fernanda; Carvalho, Bruno M; Zanotto, Tamires M; Santos, Andrey; Guadagnini, Dioze; Silva, Kelly L C; Mendes, Maria Carolina S; Rocha, Guilherme Z; Alegretti, Silmara M; Santos, Gustavo A; Catharino, Rodrigo R; Paroni, Rita; Folli, Franco; Saad, Mário José A

2018-06-01

Intestinal helminths are prevalent in individuals who live in rural areas of developing countries, where obesity, type 2 diabetes, and metabolic syndrome are rare. In the present study, we analyzed the modulation of the gut microbiota in mice infected with the helminth Strongyloides venezuelensis, and fed either a standard rodent chow diet or high-fat diet (HFD). To investigate the effects of the microbiota modulation on the metabolism, we analyzed the expression of tight-junction proteins present in the gut epithelium, inflammatory markers in the serum and tissue and quantified glucose tolerance and insulin sensitivity and resistance. Additionally, the levels of lipids related to inflammation were evaluated in the feces and serum. Our results show that infection with Strongyloides venezuelensis results in a modification of the gut microbiota, most notably by increasing Lactobacillus spp. These modifications in the microbiota alter the host metabolism by increasing the levels of anti-inflammatory cytokines, switching macrophages from a M1 to M2 pattern in the adipose tissue, increasing the expression of tight junction proteins in the intestinal cells (thereby reducing the permeability) and decreasing LPS in the serum. Taken together, these changes correlate with improved insulin signaling and sensitivity, which could also be achieved with HFD mice treated with probiotics. Additionally, helminth infected mice produce higher levels of oleic acid, which participates in anti-inflammatory pathways. These results suggest that modulation of the microbiota by helminth infection or probiotic treatment causes a reduction in subclinical inflammation, which has a positive effect on the glucose metabolism of the host. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

PubMed

Zhang, Shishuang; Zhi, Yongle; Li, Fei; Huang, Shan; Gao, Huabin; Han, Zhaoli; Ge, Xintong; Li, Dai; Chen, Fanglian; Kong, Xiaodong; Lei, Ping

2018-04-15

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aβ in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-β (Aβ) transport, and chronic cerebral hypoperfusion causes Aβ deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aβ deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aβ senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aβ clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD. Copyright © 2018 Elsevier B.V. All rights reserved.

Suppression of TNF-α and free radicals reduces systematic inflammatory and metabolic disorders: Radioprotective effects of ginseng oligopeptides on intestinal barrier function and antioxidant defense.

PubMed

He, Li-Xia; Wang, Jun-Bo; Sun, Bin; Zhao, Jian; Li, Lin; Xu, Teng; Li, Hui; Sun, Jing-Qin; Ren, Jinwei; Liu, Rui; Chen, Qi-He; Zhang, Zhao-Feng; Li, Yong

2017-02-01

Irradiation therapy is markedly associated with intestinal injure and oxidant stress. This study aimed to investigate the effects of ginseng (Panax ginseng C.A. Mey.) oligopeptides (GOP) on irradiation-induced intestinal injury and antioxidant defense in mice. BALB/c mice (8 weeks old) were randomly divided into six groups: vehicle control, irradiation control (IR), IR+whey protein [0.30 g/kg body weight (BW)], IR+GOP 0.15 g/kg BW, IR+GOP 0.30 g/kg BW and IR+GOP 0.60 g/kg BW. Postirradiation 30-day survival trial, white blood cells count and bone marrow hematopoietic system damage were performed to identify the injury degree induced by irradiation. Then, histopathology analysis was observed and intestinal permeability in vivo was quantified with fluorescein isothiocyanate-dextran. The enzyme-linked immunosorbent assay was used to determine antioxidant ability, plasma inflammatory cytokines, diamine oxidase (DAO) and endotoxin (LPS) levels. The immunohistochemistry assay was used to analyze the expression levels of tight junction proteins. We found that GOP-treated mice exhibited lower concentrations of plasma LPS and DAO and decreased instructors of inflammatory and oxidative stress which were linked to the lower intestinal permeability and higher tight junction proteins expression. The blockage of GOP was linked with the reduction of TNF-α and free radicals. The 15-day pretreatment of GOP could exhibit radioprotective effects, and another 15-day posttreatment benefited the quick repair of irradiation-induced injury. We confirm that GOP would exhibit effective therapeutic value on attenuating irradiation-induced hematopoietic, gastrointestinal and oxidative injury in cancer patients. Copyright © 2016 Elsevier Inc. All rights reserved.

Spontaneous, Immune-Mediated Gastric Inflammation in SAMP1/YitFc Mice, a Model of Crohn’s-Like Gastritis

PubMed Central

Reuter, Brian K.; Pastorelli, Luca; Brogi, Marco; Garg, Rekha R.; McBride, James A.; Rowlett, Robert M.; Arrieta, Marie C.; Wang, Xiao-Ming; Keller, Erik J.; Feldman, Sanford H.; Mize, James R.; Cominelli, Fabio; Meddings, Jonathan B.; Pizarro, Theresa T.

2011-01-01

Background & Aims Crohn’s disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn’s gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. Methods Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp. was tested in fecal pellets by PCR analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose and epithelial tight junction protein expression was measured by quantitative reverse transcription PCR analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. Results SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased in gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4+ T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. Conclusions In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn’s gastritis. PMID:21704001

Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

PubMed

Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

2016-07-05

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.

PubMed

Benedicto, Ignacio; Molina-Jiménez, Francisca; Barreiro, Olga; Maldonado-Rodríguez, Alejandra; Prieto, Jesús; Moreno-Otero, Ricardo; Aldabe, Rafael; López-Cabrera, Manuel; Majano, Pedro L

2008-10-01

Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells. We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.

Melatonin prevents secondary intra-abdominal hypertension in rats possibly through inhibition of the p38 MAPK pathway.

PubMed

Chang, Mingtao; Li, Yang; Liu, Dong; Zhang, Lianyang; Zhang, Hongguang; Tang, Hao; Zhang, Huayu

2016-08-01

Exogenous administration of melatonin has been demonstrated to down-regulate inflammatory responses and attenuate organ damage in various models. However, the salutary effect of melatonin against secondary intra-abdominal hypertension (IAH) remains unclear. This study sought to test the influence of melatonin on secondary IAH in a pathophysiological rat model and the underlying mechanisms involved. Before resuscitation, male rats underwent a combination of induced portal hypertension, applying an abdominal restraint device, and hemorrhaging to mean arterial pressure (MAP) of 40mmHg for 2h. After blood reinfusion, the rats were treated with lactated Ringer solution (LR) (30mL/h), melatonin (50mg/kg) +LR, and SB-203580 (10μmol/kg)+LR. LR was continuously infused for 6h. MAP, the inferior vena cava pressure and urine output were monitored. Histopathological examination, immunofluorescence of tight junction proteins, and transmission electron microscopy were administered. Intestinal permeability, myeloperoxidase activity, malondialdehyde, glutathione peroxidase, and levels of TNF-a, IL-2, and IL-6, were assessed. The expression of extracellular signal-regulated kinase, p38, c-Jun NH2-terminal kinase, translocation of nuclear factor kappa B subunit, signal transducers and activators of transcription and tight junction proteins were detected by Western blot. We found that melatonin inhibited the inflammatory responses, decreased expression of p38 MAPK, attenuated intestinal injury, and prevented secondary IAH. Moreover, administration of SB203580 abolished the increase in p38 MAPK and also attenuated intestinal injury. These data indicate that melatonin exerts a protective effect in intestine in secondary IAH primarily by attenuating the inflammatory responses which are in part attributable to p38 MAPK inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2006-09-01

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

Barley malt increases hindgut and portal butyric acid, modulates gene expression of gut tight junction proteins and Toll-like receptors in rats fed high-fat diets, but high advanced glycation end-products partially attenuate the effects.

PubMed

Zhong, Yadong; Teixeira, Cristina; Marungruang, Nittaya; Sae-Lim, Watina; Tareke, Eden; Andersson, Roger; Fåk, Frida; Nyman, Margareta

2015-09-01

Barley malt, a product of controlled germination, has been shown to produce high levels of butyric acid in the cecum and portal serum of rats and may therefore have anti-inflammatory effects. The aim of the study was to investigate how four barley malts, caramelized and colored malts, 50-malt and 350-malt, differing in functional characteristics concerning beta-glucan content and color, affect short-chain fatty acids (SCFA), barrier function and inflammation in the hindgut of rats fed high-fat diets. Male Wistar rats were given malt-supplemented high-fat diets for four weeks. Low and high-fat diets containing microcrystalline cellulose were incorporated as controls. All diets contained 70 g kg(-1) dietary fiber. The malt-fed groups were found to have had induced higher amounts of butyric and propionic acids in the hindgut and portal serum compared with controls, while cecal succinic acid only increased to a small extent. Fat increased the mRNA expression of tight junction proteins and Toll-like receptors (TLR) in the small intestine and distal colon of the rats, as well as the concentration of some amino acids in the portal plasma, but malt seemed to counteract these adverse effects to some extent. However, the high content of advanced glycation end-products (AGE) in caramelized malt tended to prohibit the positive effects on occludin in the small intestine and plasma amino acids seen with the other malt products. In conclusion, malting seems to be an interesting process for producing foods with positive health effects, but part of these effects may be destroyed if the malt contains a high content of AGE.

Lactobacillus reuteri increases mucus thickness and ameliorates dextran sulphate sodium-induced colitis in mice.

PubMed

Ahl, D; Liu, H; Schreiber, O; Roos, S; Phillipson, M; Holm, L

2016-08-01

The aim of this study was to investigate whether two Lactobacillus reuteri strains (rat-derived R2LC and human-derived ATCC PTA 4659 (4659)) could protect mice against colitis, as well as delineate the mechanisms behind this protection. Mice were given L. reuteri R2LC or 4659 by gavage once daily for 14 days, and colitis was induced by addition of 3% DSS (dextran sulphate sodium) to drinking water for the last 7 days of this period. The severity of disease was assessed through clinical observations, histological evaluation and ELISA measurements of myeloperoxidase (MPO) and pro-inflammatory cytokines from colonic samples. Mucus thickness was measured in vivo with micropipettes, and tight junction protein expression was assessed using immunohistochemistry. Colitis severity was significantly reduced by L. reuteri R2LC or 4659 when evaluated both clinically and histologically. The inflammation markers MPO, IL-1β, IL-6 and mKC (mouse keratinocyte chemoattractant) were increased by DSS and significantly reduced by the L. reuteri strains. The firmly adherent mucus thickness was reduced by DSS, but significantly increased by L. reuteri in both control and DSS-treated mice. Expression of the tight junction proteins occludin and ZO-1 was significantly increased in the bottom of the colonic crypts by L. reuteri R2LC. These results demonstrate that each of the two different L. reuteri strains, one human-derived and one-rat-derived, protects against colitis in mice. Mechanisms behind this protection could at least partly be explained by the increased mucus thickness as well as a tightened epithelium in the stem cell area of the crypts. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Glutamine and arginine improve permeability and tight junction protein expression in methotrexate-treated Caco-2 cells.

PubMed

Beutheu, Stéphanie; Ghouzali, Ibtissem; Galas, Ludovic; Déchelotte, Pierre; Coëffier, Moïse

2013-10-01

Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo. Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Beer Is Less Harmful for the Liver than Plain Ethanol: Studies in Male Mice Using a Binge-Drinking Model.

PubMed

Landmann, Marianne; Wagnerberger, Sabine; Kanuri, Giridhar; Ziegenhardt, Doreen; Bergheim, Ina

2015-09-01

Mechanisms involved in the less damaging effects of beer in comparison to hard spirits have not yet been fully understood. The aim of the study was to determine if the effect of beer intake on the liver differs from that of plain ethanol and if so to determine mechanisms involved. Male C57BL/6J mice received either ethanol, beer (ethanol content: 6 g/kg body weight) or iso-caloric maltodextrin solution. Markers of steatosis, lipogenesis, activation of the toll-like receptor-4 signaling cascade and lipid export in liver and tight junction proteins in duodenum were measured 6 and 12 h after acute ethanol or beer intake. Alcohol ingestion resulted in a significant increase of hepatic triglyceride accumulation 6 and 12 h after ingestion, respectively, being markedly lower in mice fed beer. Expression of sterol regulatory element-binding protein-1c mRNA was significantly lower 12 h after alcohol or beer exposure, while fatty acid synthase mRNA expression was induced in livers of ethanol-fed mice and to a lesser extent in mice fed beer 6 h after acute alcohol ingestion. Protein levels of tight junction proteins in the small intestine were similar between groups while expression of myeloid differentiation primary response gene 88 in livers was significantly induced in ethanol- but not in beer-fed mice. Concentrations of 4-hydroxynonenal protein adducts and inducible nitric oxide synthase protein were also only induced in livers of mice fed ethanol. Protein levels of apolipoprotein B were induced in livers of beer-fed mice only. Our data suggest that beer is less harmful on the development of acute alcohol-induced liver damage than plain ethanol in male mice. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

Indigenous lactobacilli strains of food and human sources reverse enteropathogenic E. coli O26:H11-induced damage in intestinal epithelial cell lines: effect on redistribution of tight junction proteins.

PubMed

Jariwala, Ruchi; Mandal, Hemanti; Bagchi, Tamishraha

2017-09-01

The aim of the study was to investigate the neutralizing effect of lactobacilli isolated from indigenous food and human sources on enteropathogenic Escherichia coli (EPEC) O26 : H11-induced epithelial barrier dysfunction in vitro. This was assessed by transepithelial electrical resistance (TEER) and permeability assays using intestinal cell lines, HT-29 and Caco-2. Furthermore, the expression and distribution of tight junction (TJ) proteins were analysed by qRT-PCR and immunofluorescence assay, respectively. The nine strains used in the study were from different species viz. Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus salivarius and Lactobacillus plantarum. All strains were able to reverse the decrease in TEER and corresponding increase in permeability across E. coli-infected monolayers. Maximum reversal was observed after 18 h [up to 93.8±2.0 % by L. rhamnosus GG followed by L. fermentum IIs11.2 (92.6±2.2 %) and L. plantarum GRI-2 (91.9±0.9 %)] of lactobacilli exposure following EPEC O26 : H11 infection. All strains were able to redistribute the TJ proteins to the cell periphery either partially or completely. Moreover, L. helveticus FA-7 was also able to significantly increase the mRNA expression of ZO-1 and claudin-1 (2.5-fold and 3.0-fold, respectively; P<0.05). The rapid reversal observed by these strains could be mostly because of the redistribution rather than increased mRNA expression of TJ proteins. In conclusion, L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 were good in all the aspects studied, and the other strains were good in some aspects. L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 can therefore be used for potential therapeutic purpose against intestinal epithelial dysfunction.

Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis.

PubMed

Matsumoto, Kotaro; Ichimura, Mayuko; Tsuneyama, Koichi; Moritoki, Yuki; Tsunashima, Hiromichi; Omagari, Katsuhisa; Hara, Masumi; Yasuda, Ichiro; Miyakawa, Hiroshi; Kikuchi, Kentaro

2017-01-01

Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.

EICOSAPENTAENOIC ACID ENHANCES HEATSTROKE-IMPAIRED INTESTINAL EPITHELIAL BARRIER FUNCTION IN RATS.

PubMed

Xiao, Guizhen; Yuan, Fangfang; Geng, Yan; Qiu, Xiaowen; Liu, Zhifeng; Lu, Jiefu; Tang, Liqun; Zhang, Yali; Su, Lei

2015-10-01

Dysfunction of the intestinal barrier plays an important role in the pathological process of heatstroke. Omega-3 (or n-3) polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), help protect the intestinal mucosal barrier. This study assessed if pretreating rats with EPA or DHA could alleviate heat stress-induced damage to the intestinal barrier caused by experimental heatstroke. Male Wistar rats were pregavaged with either EPA, DHA, corn oil, or normal saline (all 1 g/kg) for 21 days before the heatstroke experiment (control rats were not exposed to heat). Experimental rats were exposed to an ambient temperature of 37°C and 60% humidity to induce heatstroke, and then they were allowed to recover at room temperature after rapid cooling. Survival time of rats was monitored after heatstroke. Horseradish peroxidase flux from the gut lumen and the level of plasma D-lactate were measured to analyze intestinal permeability at 6 h after heatstroke. Plasma endotoxin levels were determined using a limulus amoebocyte lysate assay. Expressions of the tight junction (TJ) proteins occludin and ZO-1 were analyzed by Western blot and localized by immunofluorescence microscopy. Tight junction protein morphology was observed by transmission electron microscopy. Fatty acids of ileal mucosa were analyzed using gas chromatography-mass selective detector. Eicosapentaenoic acid significantly increased survival time after heatstroke. Eicosapentaenoic acid significantly decreased intestinal permeability and plasma endotoxin levels. Eicosapentaenoic acid effectively attenuated the heatstroke-induced disruption of the intestinal structure and improved the histology score, whereas DHA was less effective, and corn oil was ineffective. Pretreatment with EPA also increased expression of occludin and ZO-1 to effectively prevent TJ disruption. Eicosapentaenoic acid pretreatment enriched itself in the membrane of intestinal cells. Our results indicate that EPA pretreatment is more effective than DHA pretreatment in attenuating heat-induced intestinal dysfunction and preventing TJ damage. Enhanced expression of TJ proteins that support the epithelial barrier integrity may be important for maintaining a functional intestinal barrier during heatstroke.

Chronic depletion of gonadal testosterone leads to blood-brain barrier dysfunction and inflammation in male mice.

PubMed

Atallah, Afnan; Mhaouty-Kodja, Sakina; Grange-Messent, Valérie

2017-09-01

A dysfunction in the blood-brain barrier (BBB) is associated with many neurological and metabolic disorders. Although sex steroid hormones have been shown to impact vascular tone, endothelial function, oxidative stress, and inflammatory responses, there are still no data on the role of testosterone in the regulation of BBB structure and function. In this context, we investigated the effects of gonadal testosterone depletion on the integrity of capillary BBB and the surrounding parenchyma in male mice. Our results show increased BBB permeability for different tracers and endogenous immunoglobulins in chronically testosterone-depleted male mice. These results were associated with disorganization of tight junction structures shown by electron tomography and a lower amount of tight junction proteins such as claudin-5 and ZO-1. BBB leakage was also accompanied by activation of astrocytes and microglia, and up-regulation of inflammatory molecules such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin 1 beta (IL-1β), and tumor necrosis factor (TNF). Supplementation of castrated male mice with testosterone restored BBB selective permeability, tight junction integrity, and almost completely abrogated the inflammatory features. The present demonstration that testosterone transiently impacts cerebrovascular physiology in adult male mice should help gain new insights into neurological and metabolic diseases linked to hypogonadism in men of all ages.

Crumbs3 Is Essential for Proper Epithelial Development and Viability

PubMed Central

Whiteman, Eileen L.; Fan, Shuling; Harder, Jennifer L.; Walton, Katherine D.; Liu, Chia-Jen; Soofi, Abdul; Fogg, Vanessa C.; Hershenson, Marc B.; Dressler, Gregory R.; Deutsch, Gail H.; Gumucio, Deborah L.

2014-01-01

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton. PMID:24164893

Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.

PubMed

Yang, Mei; Cong, Min; Peng, Xiuming; Wu, Junrui; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing

2016-05-18

Milk fat globule membrane (MFGM) proteins have many functions. To explore the different proteomics of human and bovine MFGM, MFGM proteins were separated from human and bovine colostrum and mature milk, and analyzed by the iTRAQ proteomic approach. A total of 411 proteins were recognized and quantified. Among these, 232 kinds of differentially expressed proteins were identified. These differentially expressed proteins were analyzed based on multivariate analysis, gene ontology (GO) annotation and KEGG pathway. Biological processes involved were response to stimulus, localization, establishment of localization, and the immune system process. Cellular components engaged were the extracellular space, extracellular region parts, cell fractions, and vesicles. Molecular functions touched upon were protein binding, nucleotide binding, and enzyme inhibitor activity. The KEGG pathway analysis showed several pathways, including regulation of the actin cytoskeleton, focal adhesion, neurotrophin signaling pathway, leukocyte transendothelial migration, tight junction, complement and coagulation cascades, vascular endothelial growth factor signaling pathway, and adherens junction. These results enhance our understanding of different proteomes of human and bovine MFGM across different lactation phases, which could provide important information and potential directions for the infant milk powder and functional food industries.

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

PubMed Central

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

2009-01-01

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

Effects of electromagnetic pulse irradiation on the mouse blood-testicle barrier.

PubMed

Hou, Wu-Gang; Zhao, Jie; Li, Zhen; Li, Wei; Li, Teng; Xiong, Li-Ze; Zhang, Yuan-Qiang

2012-07-01

To investigate the effects of electromagnetic pulse irradiation on the mouse blood-testicle barrier (BTB) and spermatogenesis. After whole body irradiation with 400 kV/m electromagnetic pulse irradiation, the mouse testicles and BTB permeability were observed using hematoxylin-eosin, Evans blue, and lanthanum nitrate as tracers. The expression of the BTB tight junction protein occludin was examined using real-time polymerase chain reaction and Western blotting. At 1, 7, and 14 days after irradiation, the BTB structure was damaged, the BTB permeability was significantly increased, numerous apoptotic or necrotic spermatogenic cells were found in the lumen, and the mRNA and protein expression levels of occludin were markedly decreased. The BTB structure and occludin expression levels had gradually recovered by 21 and 28 days after irradiation. Electromagnetic pulse irradiation damaged the structure and function of mouse BTB, resulting in apoptosis or necrosis of the spermatogenic cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE) Based Diagnosis and Therapy

PubMed Central

English, Diana P.; Santin, Alessandro D.

2013-01-01

Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents. PMID:23685873

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

PubMed Central

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

2014-01-01

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

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

PubMed

Sun, Xiaofei; Park, Craig B; Deng, Wenbo; Potter, S Steven; Dey, Sudhansu K

2016-04-01

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. © FASEB.

Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation

PubMed Central

Sun, Xiaofei; Park, Craig B.; Deng, Wenbo; Potter, S. Steven; Dey, Sudhansu K.

2016-01-01

Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion of Msx genes (Msx1d/d/Msx2d/d) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx’s roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type–specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection from Msx1d/d/Msx2d/d and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated in Msx1d/d/Msx2d/d uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) in Msx1f/f/Msx2f/f females; the patterns were lost in Msx1d/d/Msx2d/d epithelia on d 5, suggesting important roles during implantation. The results suggest that Msx genes play important roles during uterine receptivity including modulation of epithelial junctional activity.—Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. PMID:26667042

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

PubMed

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

2016-12-01

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

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

PubMed Central

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

2009-01-01

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

Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

PubMed

Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

2016-12-01

Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

Ghosh, Pradipta

2017-01-01

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

Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis

PubMed Central

Saint-Laurent, Olivia; Bourbonnière, Lyne; Larouche, Sandra; Larochelle, Catherine; Michel, Laure; Charabati, Marc; Abadier, Michael; Zandee, Stephanie; Haghayegh Jahromi, Neda; Gowing, Elizabeth; Pittet, Camille; Lyck, Ruth; Engelhardt, Britta

2017-01-01

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood–brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35–55–induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity. PMID:28069965

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion.

PubMed

Baum, Jake; Tonkin, Christopher J; Paul, Aditya S; Rug, Melanie; Smith, Brian J; Gould, Sven B; Richard, Dave; Pollard, Thomas D; Cowman, Alan F

2008-03-13

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.

Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.

PubMed

Huang, Ching-Wen; Chen, Huai-Yi; Yen, Meng-Hua; Chen, Jeremy J W; Young, Tai-Horng; Cheng, Ji-Yen

2011-01-01

Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study. A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

Intestinal Fluid Permeability in Atlantic Salmon (Salmo salar L.) Is Affected by Dietary Protein Source.

PubMed

Hu, Haibin; Kortner, Trond M; Gajardo, Karina; Chikwati, Elvis; Tinsley, John; Krogdahl, Åshild

2016-01-01

In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingredients can increase fecal water content creating a diarrhea-like condition which may impair gut function and reduce fish growth. The present study aimed to strengthen understanding of the underlying mechanisms by observing effects of various alternative plant protein sources when replacing fish meal on expression of genes encoding proteins playing key roles in regulation of water transport across the mucosa of the distal intestine (DI). A 48-day feeding trial was conducted with five diets: A reference diet (FM) in which fish meal (72%) was the only protein source; Diet SBMWG with a mix of soybean meal (30%) and wheat gluten (22%); Diet SPCPM with a mix of soy protein concentrate (30%) and poultry meal (6%); Diet GMWG with guar meal (30%) and wheat gluten (14.5%); Diet PM with 58% poultry meal. Compared to fish fed the FM reference diet, fish fed the soybean meal containing diet (SBMWG) showed signs of enteritis in the DI, increased fecal water content of DI chyme and higher plasma osmolality. Altered DI expression of a battery of genes encoding aquaporins, ion transporters, tight junction and adherens junction proteins suggested reduced transcellular transport of water as well as a tightening of the junction barrier in fish fed the SBMWG diet, which may explain the observed higher fecal water content and plasma osmolality. DI structure was not altered for fish fed the other experimental diets but alterations in target gene expression and fecal water content were observed, indicating that alterations in water transport components may take place without clear effects on intestinal structure.

A permeability barrier surrounds taste buds in lingual epithelia

PubMed Central

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

2014-01-01

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

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

PubMed Central

Fan, Jie; Cai, Bin; Zeng, Min; Hao, Yanyan

2015-01-01

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium. PMID:21556948

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

PubMed

Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

2018-01-01

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

[Peripheral neuropathy and blood-nerve barrier].

PubMed

Kanda, Takashi

2009-11-01

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

The ginger component 6-shogaol prevents TNF-α-induced barrier loss via inhibition of PI3K/Akt and NF-κB signaling.

PubMed

Luettig, Julia; Rosenthal, Rita; Lee, In-Fah M; Krug, Susanne M; Schulzke, Jörg D

2016-12-01

Anti-inflammatory properties of the ginger-derived pungent component 6-shogaol (6-SG) have been studied intensively in recent years. Purpose of this study was to characterize the influence of 6-SG on inflammation-related intestinal barrier dysfunction, especially its paracellular component. The effect of 6-SG was studied in the human intestinal cell models HT-29/B6 and Caco-2 either under control conditions or challenged by the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Electrophysiological measurements, freeze-fracture electron microscopy, and protein analyses were performed. 6-SG partially prevented both, the TNF-α-induced decrease in transepithelial resistance and the rise in fluorescein permeability. By inhibiting phosphatidylinositol-3-kinase/Akt signaling 6-SG prevented the TNF-α-induced increase in protein expression of claudin-2, a channel-forming tight junction protein. In addition, the TNF-α-induced disassembly of the sealing tight junction protein claudin-1 was attenuated, the latter of which was due to TNF-α-triggered phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB). 6-SG has barrier-protective effects by affecting TNF-α-induced claudin-2 upregulation and claudin-1 disassembly via inhibition of phoshatidylinositol-3-kinase/Akt and nuclear factor kappa light chain enhancer of activated B-cell signaling. Therefore, 6-SG-containing food might be beneficial for barrier preservation during intestinal inflammation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Claudin-19 and the Barrier Properties of the Human Retinal Pigment Epithelium

PubMed Central

Peng, Shaomin; Rao, Veena S.; Adelman, Ron A.

2011-01-01

Purpose. The retinal pigment epithelium (RPE) separates photoreceptors from choroidal capillaries, but in age-related macular degeneration (AMD) capillaries breach the RPE barrier. Little is known about human RPE tight junctions or the effects of serum on the retinal side of the RPE. Methods. Cultured human fetal RPE (hfRPE) was assessed by the transepithelial electrical resistance (TER) and the transepithelial diffusion of methylated polyethylene glycol (mPEG). Claudins and occludin were monitored by quantitative RT-PCR, immunoblotting, and immunofluorescence. Results. Similar to freshly isolated hfRPE, claudin-19 mRNA was 25 times more abundant than claudin-3. Other detectable claudin mRNAs were found in even lesser amounts, as little as 3000 times less abundant than claudin-19. Claudin-1 and claudin-10b were detected only in subpopulations of cells, whereas others were undetectable. Knockdown of claudin-19 by small interfering RNA (siRNA) eliminated the TER. siRNAs for other claudins had minimal effects. Serum affected tight junctions only when presented to the retinal side of the RPE. The TER increased 2 times, and the conductance of K+ relative to Na+ decreased without affecting the permeability of mPEG. These effects correlated with increased steady-state levels of occludin. Conclusions. Fetal human RPE is a claudin-19–dominant epithelium that has regional variations in claudin-expression. Apical serum decreases RPE permeability, which might be a defense mechanism that would retard the spread of edema due to AMD. PMID:21071746

Hydrocephalus due to multiple ependymal malformations is caused by mutations in the MPDZ gene.

PubMed

Saugier-Veber, Pascale; Marguet, Florent; Lecoquierre, François; Adle-Biassette, Homa; Guimiot, Fabien; Cipriani, Sara; Patrier, Sophie; Brasseur-Daudruy, Marie; Goldenberg, Alice; Layet, Valérie; Capri, Yline; Gérard, Marion; Frébourg, Thierry; Laquerrière, Annie

2017-05-01

Congenital hydrocephalus is considered as either acquired due to haemorrhage, infection or neoplasia or as of developmental nature and is divided into two subgroups, communicating and obstructive. Congenital hydrocephalus is either syndromic or non-syndromic, and in the latter no cause is found in more than half of the patients. In patients with isolated hydrocephalus, L1CAM mutations represent the most common aetiology. More recently, a founder mutation has also been reported in the MPDZ gene in foetuses presenting massive hydrocephalus, but the neuropathology remains unknown. We describe here three novel homozygous null mutations in the MPDZ gene in foetuses whose post-mortem examination has revealed a homogeneous phenotype characterized by multiple ependymal malformations along the aqueduct of Sylvius, the third and fourth ventricles as well as the central canal of the medulla, consisting in multifocal rosettes with immature cell accumulation in the vicinity of ependymal lining early detached from the ventricular zone. MPDZ also named MUPP1 is an essential component of tight junctions which are expressed from early brain development in the choroid plexuses and ependyma. Alterations in the formation of tight junctions within the ependyma very likely account for the lesions observed and highlight for the first time that primary multifocal ependymal malformations of the ventricular system is genetically determined in humans. Therefore, MPDZ sequencing should be performed when neuropathological examination reveals multifocal ependymal rosette formation within the aqueduct of Sylvius, of the third and fourth ventricles and of the central canal of the medulla.

Claudins in teleost fishes

PubMed Central

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

2013-01-01

Teleost fishes are a large and diverse animal group that represent close to 50% of all described vertebrate species. This review consolidates what is known about the claudin (Cldn) family of tight junction (TJ) proteins in teleosts. Cldns are transmembrane proteins of the vertebrate epithelial/endothelial TJ complex that largely determine TJ permeability. Cldns achieve this by expressing barrier or pore forming properties and by exhibiting distinct tissue distribution patterns. So far, ~63 genes encoding for Cldn TJ proteins have been reported in 16 teleost species. Collectively, cldns (or Cldns) are found in a broad array of teleost fish tissues, but select genes exhibit restricted expression patterns. Evidence to date strongly supports the view that Cldns play a vital role in the embryonic development of teleost fishes and in the physiology of tissues and organ systems studied thus far. PMID:24665402

Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.

PubMed

Ohtsuki, Sumio; Hirayama, Mio; Ito, Shingo; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2014-06-01

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.

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

PubMed

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

2018-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2012-01-01

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

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

PubMed Central

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

2018-01-01

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

Expression of P-aPKC-iota, E-cadherin, and beta-catenin related to invasion and metastasis in hepatocellular carcinoma.

PubMed

Du, Guang-Sheng; Wang, Jian-Ming; Lu, Jin-Xi; Li, Qiang; Ma, Chao-Qun; Du, Ji-Tao; Zou, Sheng-Quan

2009-06-01

Atypical protein kinase C iota (aPKC-iota) and its associated intracellular molecules, E-cadherin and beta-catenin, are important for cell polarization in tumorigenesis and progression. Expression of aPKC-iota, P-aPKC-iota (activated aPKC-iota), E-cadherin, and beta-catenin in hepatocellular carcinoma (HCC) was measured, and correlation with clinicopathological characteristics of HCC was analyzed. Paraffin-embedded tumor tissue was obtained from patients with HCC after resection without preoperative radiotherapy or chemotherapy. Gene expression was detected by polymerase chain reaction (PCR), and protein expression was detected by immunohistochemistry and Western blot analysis. Expressions of aPKC-iota, P-aPKC-iota, E-cadherin, and beta-catenin were analyzed with relation to the clinicopathological data. The gene and protein expression of aPKC-iota are obviously higher in HCC tissues than that in peritumoral tissues and normal tissues by semiquantitative PCR and immunohistochemistry methods. Accumulation of aPKC-iota in HCC cytoplasm and nucleolus inhibited the later formation of belt-like adherens junctions (AJs) and/or tight junctions (TJs) in cell-cell contact. E-cadherin was reduced and accumulation of cytoplasm beta-catenin was increased in HCC. The expression of aPKC-iota was closely related to pathological differentiation, tumor size, invasion, and metastasis of HCC. Accumulation of cytoplasm aPKC-iota may reflect pathological differentiation, invasion, and metastasis potential of HCC. In this regard, our study on HCC revealed the potential usefulness of aPKC-iota, E-cadherin, and beta-catenin as a prognostic marker, closely related to pathological differentiation, invasion, metastasis, and prognosis of HCC.

Arsenic-induced cutaneous hyperplastic lesions are associated with the dysregulation of Yap, a Hippo signaling-related protein

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

Li, Changzhao; Srivastava, Ritesh K.; Elmets, Craig A.

2013-09-06

Highlights: •Arsenic activates canonical Hippo signaling pathway and up-regulates αCatenin in the skin. •Arsenic activates transcriptional activity of Yap by its nuclear translocation. •Yap is involved in the disruption of tight/adherens junctions in arsenic-exposed animals. -- Abstract: Arsenic exposure in humans causes a number of toxic manifestations in the skin including cutaneous neoplasm. However, the mechanism of these alterations remains elusive. Here, we provide novel observations that arsenic induced Hippo signaling pathway in the murine skin. This pathway plays crucial roles in determining organ size during the embryonic development and if aberrantly activated in adults, contributes to the pathogenesis ofmore » epithelial neoplasm. Arsenic treatment enhanced phosphorylation-dependent activation of LATS1 kinase and other Hippo signaling regulatory proteins Sav1 and MOB1. Phospho-LATS kinase is known to catalyze the inactivation of a transcriptional co-activator, Yap. However, in arsenic-treated epidermis, we did not observed its inactivation. Thus, as expected, unphosphorylated-Yap was translocated to the nucleus in arsenic-treated epidermis. Yap by binding to the transcription factors TEADs induces transcription of its target genes. Consistently, an up-regulation of Yap-dependent target genes Cyr61, Gli2, Ankrd1 and Ctgf was observed in the skin of arsenic-treated mice. Phosphorylated Yap is important in regulating tight and adherens junctions through its binding to αCatenin. We found disruption of these junctions in the arsenic-treated mouse skin despite an increase in αCatenin. These data provide evidence that arsenic-induced canonical Hippo signaling pathway and Yap-mediated disruption of tight and adherens junctions are independently regulated. These effects together may contribute to the carcinogenic effects of arsenic in the skin.« less

Multi-orbit tight binding calculations for spin transfer torque in magnetic tunneling junctions

NASA Astrophysics Data System (ADS)

You, Chun-Yeol; Han, Jae-Ho; Lee, Hyun-Woo

2012-04-01

We investigate the spin transfer torque (STT) with multi-orbit tight binding model in the magnetic tunneling junctions (MTJs). So far, most of the theoretical works based on the non-equilibrium Keldysh Green's function method employ a single band model for the simplicity, except a few first principle studies. Even though the single band model captures main physics of STT in MTJ, multi-band calculation reveals new features of the STT that depend on band parameters, such as insulator bandgap, inter-band hopping energy of the ferromagnetic layer. We find that the sign change of perpendicular torkance with bandgap of the insulator layer, and when we allow the inter-band hopping, the bias dependences of perpendicular STT are dramatically changed, while no noticeable changes in parallel STT are found.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R.

PubMed

Cai, Siwei; Yang, Qianhui; Hou, Mengzhu; Han, Qian; Zhang, Hanyu; Wang, Jiantao; Qi, Chen; Bo, Qiyu; Ru, Yusha; Yang, Wei; Gu, Zhongxiu; Wei, Ruihua; Cao, Yunshan; Li, Xiaorong; Zhang, Yan

2018-01-01

Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH's anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH's protection. Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested partial rescue of functional defects by α-MSH in diabetic retinas; and H&E staining revealed significantly increased thickness of all layers in α-MSH-treated diabetic retinas. Mechanistically, α-MSH corrected aberrant transcript and protein expression of pro-inflammatory factor and tight junction genes in the diseased retinas; moreover, it prevented abnormal changes in TEER and permeability in HG-stimulated RF6A cells, and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist specific to MC4R. This study showed previously undescribed protective effects of α-MSH on inhibiting BRB breakdown and vascular leakage, improving electrophysiological functions and morphology in early diabetic retinas, which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions. α-MSH acts predominantly on MC4R to antagonize hyperpermeability in retinal microvessel endothelial cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

Phase transition in one Josephson junction with a side-coupled magnetic impurity

NASA Astrophysics Data System (ADS)

Zhi, Li-Ming; Wang, Xiao-Qi; Jiang, Cui; Yi, Guang-Yu; Gong, Wei-Jiang

2018-04-01

This work focuses on one Josephson junction with a side-coupled magnetic impurity. And then, the Josephson phase transition is theoretically investigated, with the help of the exact diagonalization approach. It is found that even in the absence of intradot Coulomb interaction, the magnetic impurity can efficiently induce the phenomenon of Josephson phase transition, which is tightly related to the spin correlation manners (i.e., ferromagnetic or antiferromagnetic) between the impurity and the junction. Moreover, the impurity plays different roles when it couples to the dot and superconductor, respectively. This work can be helpful in describing the influence of one magnetic impurity on the supercurrent through the Josephson junction.

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

PubMed

Ohno, Kinji; Rahman, Mohammad Alinoor; Nazim, Mohammad; Nasrin, Farhana; Lin, Yingni; Takeda, Jun-Ichi; Masuda, Akio

2017-08-01

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChE T , AChE H , and AChE R are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

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

NASA Astrophysics Data System (ADS)

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

2011-08-01

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

Combining systems pharmacology, transcriptomics, proteomics, and metabolomics to dissect the therapeutic mechanism of Chinese herbal Bufei Jianpi formula for application to COPD

PubMed Central

Zhao, Peng; Yang, Liping; Li, Jiansheng; Li, Ya; Tian, Yange; Li, Suyun

2016-01-01

Bufei Jianpi formula (BJF) has long been used as a therapeutic agent in the treatment of COPD. Systems pharmacology identified 145 active compounds and 175 potential targets of BJF in a previous study. Additionally, BJF was previously shown to effectively prevent COPD and its comorbidities, such as ventricular hypertrophy, by inhibition of inflammatory cytokine production, matrix metalloproteinases expression, and other cytokine production, in vivo. However, the system-level mechanism of BJF for the treatment of COPD is still unclear. The aim of this study was to gain insight into its system-level mechanisms by integrating transcriptomics, proteomics, and metabolomics together with systems pharmacology datasets. Using molecular function, pathway, and network analyses, the genes and proteins regulated in COPD rats and BJF-treated rats could be mainly attributed to oxidoreductase activity, antioxidant activity, focal adhesion, tight junction, or adherens junction. Furthermore, a comprehensive analysis of systems pharmacology, transcript, protein, and metabolite datasets is performed. The results showed that a number of genes, proteins, metabolites regulated in BJF-treated rats and potential target proteins of BJF were involved in lipid metabolism, cell junction, oxidative stress, and inflammatory response, which might be the system-level therapeutic mechanism of BJF treatment. PMID:27042044

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

Osmosis in Cortical Collecting Tubules

PubMed Central

Schafer, James A.; Troutman, Susan L.; Andreoli, Thomas E.

1974-01-01

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s–1) Pfl→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10–4 (SEM); the value of Pfb→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10–4 cm s–1. We also measured apparent urea permeability coefficients (cm s–1) from 14C-urea fluxes from lumen to bath (P DDurea l→b) and from bath to lumen (P DDurea b→l). For hypotonic luminal solutions and isotonic bathing solutions, P DDurea l→b was 0.045 ± 0.004 x 10–4 and was unaffected by ADH. The ADH-independent values of P DDurea l→b and P urea b→l were, respectively, 0.216 ± 0.022 x 10–4 cm s–1 and 0.033 ± 0.002 x 10–4 cm s–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P DDurea l→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of Pfb→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of Pfl→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes. PMID:4846768

Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis.

PubMed

Pradhan-Sundd, Tirthadipa; Zhou, Lili; Vats, Ravi; Jiang, An; Molina, Laura; Singh, Sucha; Poddar, Minakshi; Russell, Jacquelyn; Stolz, Donna B; Oertel, Michael; Apte, Udayan; Watkins, Simon; Ranganathan, Sarangarajan; Nejak-Bowen, Kari N; Sundd, Prithu; Monga, Satdarshan Pal

2018-06-01

β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro. We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337). © 2017 by the American Association for the Study of Liver Diseases.

Human immunodeficiency virus-associated disruption of mucosal barriers and its role in HIV transmission and pathogenesis of HIV/AIDS disease

PubMed Central

Tugizov, Sharof

2016-01-01

Abstract Oral, intestinal and genital mucosal epithelia have a barrier function to prevent paracellular penetration by viral, bacterial and other pathogens, including human immunodeficiency virus (HIV). HIV can overcome these barriers by disrupting the tight and adherens junctions of mucosal epithelia. HIV-associated disruption of epithelial junctions may also facilitate paracellular penetration and dissemination of other viral pathogens. This review focuses on possible molecular mechanisms of HIV-associated disruption of mucosal epithelial junctions and its role in HIV transmission and pathogenesis of HIV and acquired immune deficiency syndrome (AIDS). PMID:27583187

A permeability barrier surrounds taste buds in lingual epithelia.

PubMed

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

2015-01-01

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

Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

PubMed Central

Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

2010-01-01

AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

Safety, efficacy, and molecular mechanism of claudin-1-specific peptides to enhance blood-nerve-barrier permeability.

PubMed

Sauer, Reine-Solange; Krug, Susanne M; Hackel, Dagmar; Staat, Christian; Konasin, Natalia; Yang, Shaobing; Niedermirtl, Benedikt; Bosten, Judith; Günther, Ramona; Dabrowski, Sebastian; Doppler, Kathrin; Sommer, Claudia; Blasig, Ingolf E; Brack, Alexander; Rittner, Heike L

2014-07-10

The blood-nerve barrier consists of the perineurium and endoneurial vessels. The perineurial barrier is composed of a basal membrane and a layer of perineurial cells sealed by tight junction proteins preventing e.g. application of analgesics for selective regional pain control. One of the barrier-sealing proteins in the blood-nerve barrier is claudin-1. Therefore, the claudin-1-peptidomimetics (C1C2), derived from the first extracellular loop (ECL1) on claudin-1 was developed. In this study, we further evaluated the expression of tight junction proteins in the perineurium in Wistar rats and characterized the specificity, in vivo applicability, mechanism of action as well as the biocompatibility of C1C2. In the perineurium, claudin-19, tricellulin and ZO-1, but no claudin-2, 3, 8 and -11 were expressed. C1C2 specifically bound to the ECL1 of claudin-1 and fluorescent 5,6-carboxytetramethylrhodamine-C1C2 was rapidly internalized. Opening the perineurium with C1C2 reduced the mRNA and protein expression of claudin-1 and increased small and macromolecule permeability into the peripheral nerve. Application of C1C2 facilitated regional analgesia using μ-opioid receptor agonists like DAMGO or morphine without motor impairment in naïve rats as well as rats with hind paw inflammation. In contrast the control peptide C2C2 derived from ECL1 on claudin-2 did neither open the barrier nor facilitated opioid-mediated regional analgesia. C1C2 delivery was well tolerated and caused no morphological and functional nerve damage. C1C2 effects could be reversed by interference with the wnt-signal-transduction pathway, specifically the homeobox transcription factor cdx2, using a glycogen-synthase-kinase-3 inhibitor. In summary, we describe the composition of and a pathway to open the perineurial barrier employing a peptide to deliver hydrophilic substances to the peripheral nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice

PubMed Central

Wu, Alex G.; Jaja-Chimedza, Asha; Graf, Brittany L.; Waterman, Carrie; Verzi, Michael P.; Raskin, Ilya

2017-01-01

Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers and increasing tight-junction proteins. This effect is consistent with Nrf2-mediated anti-inflammatory/antioxidant signaling pathway documented for other isothiocyanates similar to MIC-1. Therefore, MSE, enriched with MIC-1, may be useful in prevention and treatment of UC. PMID:28922365

Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice.

PubMed

Kim, Youjin; Wu, Alex G; Jaja-Chimedza, Asha; Graf, Brittany L; Waterman, Carrie; Verzi, Michael P; Raskin, Ilya

2017-01-01

Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers and increasing tight-junction proteins. This effect is consistent with Nrf2-mediated anti-inflammatory/antioxidant signaling pathway documented for other isothiocyanates similar to MIC-1. Therefore, MSE, enriched with MIC-1, may be useful in prevention and treatment of UC.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

PubMed

Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

PubMed Central

Benedicto, Ignacio; Lehmann, Guillermo L.; Ginsberg, Michael; Nolan, Daniel J.; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M.; Prusky, Glen T.; Llanos, Pierre; Rabbany, Sina Y.; Maminishkis, Arvydas; Miller, Sheldon S.; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-01-01

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease. PMID:28524846

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis

PubMed Central

Feng, Qing-Yang; Hu, Zi-Xuan; Song, Xi-Ling; Pan, Hong-Wei

2017-01-01

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy. PMID:28730091

Targeting blood–brain barrier changes during inflammatory pain: an opportunity for optimizing CNS drug delivery

PubMed Central

Ronaldson, Patrick T; Davis, Thomas P

2012-01-01

The blood–brain barrier (BBB) is the most significant obstacle to effective CNS drug delivery. It possesses structural and biochemical features (i.e., tight-junction protein complexes and, influx and efflux transporters) that restrict xenobiotic permeation. Pathophysiological stressors (i.e., peripheral inflammatory pain) can alter BBB tight junctions and transporters, which leads to drug-permeation changes. This is especially critical for opioids, which require precise CNS concentrations to be safe and effective analgesics. Recent studies have identified molecular targets (i.e., endogenous transporters and intracellular signaling systems) that can be exploited for optimization of CNS drug delivery. This article summarizes current knowledge in this area and emphasizes those targets that present the greatest opportunity for controlling drug permeation and/or drug transport across the BBB in an effort to achieve optimal CNS opioid delivery. PMID:22468221

Gap junction plasticity as a mechanism to regulate network-wide oscillations

PubMed Central

Nicola, Wilten; Clopath, Claudia

2018-01-01

Cortical oscillations are thought to be involved in many cognitive functions and processes. Several mechanisms have been proposed to regulate oscillations. One prominent but understudied mechanism is gap junction coupling. Gap junctions are ubiquitous in cortex between GABAergic interneurons. Moreover, recent experiments indicate their strength can be modified in an activity-dependent manner, similar to chemical synapses. We hypothesized that activity-dependent gap junction plasticity acts as a mechanism to regulate oscillations in the cortex. We developed a computational model of gap junction plasticity in a recurrent cortical network based on recent experimental findings. We showed that gap junction plasticity can serve as a homeostatic mechanism for oscillations by maintaining a tight balance between two network states: asynchronous irregular activity and synchronized oscillations. This homeostatic mechanism allows for robust communication between neuronal assemblies through two different mechanisms: transient oscillations and frequency modulation. This implies a direct functional role for gap junction plasticity in information transmission in cortex. PMID:29529034

Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model.

PubMed

Rubio-Araiz, Ana; Porcu, Francesca; Pérez-Hernández, Mercedes; García-Gutiérrez, Mª Salud; Aracil-Fernández, María Auxiliadora; Gutierrez-López, María Dolores; Guerri, Consuelo; Manzanares, Jorge; O'Shea, Esther; Colado, María Isabel

2017-07-01

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood-brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4-knockout mice under a binge-like EtOH drinking protocol. Immunohistochemical studies showed reduced immunoreactivity of the basal lamina protein, collagen-IV and of the tight junction protein, claudin-5 in dorsolateral prefrontal cortex of alcoholics. There was also increased MMP-9 activity and expression of phosphorylated ERK1/2 and p-38. Greater number of CD45+ IR cells were observed associated with an enhanced neuroinflammatory response reflected by increased GFAP and Iba-1 immunostaining. To further explore effects of high EtOH consumption on BBB integrity we studied TLR4-knockout mice exposed to the drinking in the dark paradigm. Repetitive EtOH exposure in wild-type mice decreased hippocampal expression of laminin and collagen-IV and increased IgG immunoreactivity, indicating IgG extravasation. Western blot analysis also revealed increased MyD88 and p-ERK1/2 levels. None of these changes was observed in TLR4-knockout mice. Collectively, these findings indicate that chronic EtOH increases degradation of tight junctions and extracellular matrix in postmortem human brain and induces a neuroinflammatory response associated with activation of ERK1/2 and p-38 and greater MMP-9 activity. The EtOH-induced effects on BBB impairment are not evident in the hippocampus of TLR4-knockout mice, suggesting the involvement of TLR4 signaling in the underlying mechanism leading to BBB disruption in mice. © 2016 Society for the Study of Addiction.

Variable expression of podocyte-related markers in the glomeruloid bodies in Wilms tumor.

PubMed

Kanemoto, Katsuyoshi; Takahashi, Shori; Shu, Yujing; Usui, Joichi; Tomari, Shinsuke; Yan, Kunimasa; Hamazaki, Yutaka; Nagata, Michio

2003-09-01

Several podocyte-related markers are organized to express in glomerular differentiation. However, whether expression of them is virtually synchronized and a reliable indicator of the state of differentiation is unknown. The present study investigated, by immunohistochemistry, the divergent expression of several podocyte markers in the improperly differentiated glomeruloid bodies from four cases of Wilms tumors. The glomeruloid bodies were classified into immature (IGB) or mature forms (MGB) based on morphology and epithelial features. Podocytes in IGB expressed WT1, synaptopodin, podocalyxin, and nephrin, and their expression was stronger in MGB. In contrast, Pax2 was strong in IGB and diminished in MGB. p27 was first expressed in MGB. The expression pattern in each molecule mimics normal glomerulogenesis. Podocytes in MGB showed persistent expression of bcl-2 and cytokeratin with synaptopodin, podocalyxin, and nephrin by serial section, a finding unusual for normal glomerulogenesis. Moreover, parietal cells in MGB also occasionally expressed these podocyte markers. The ultrastructure revealed that podocytes in MGB showed tight junctions without foot process formations, which indicated incomplete differentiation. These results suggest that a set of podocyte differentiation markers are occasionally diversely expressed, and raise the possibility that expression of these markers is insufficient to determine the state of terminal differentiation in podocytes.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

PubMed

de Paiva, Rita Marcia Cardoso; Grazielle-Silva, Viviane; Cardoso, Mariana Santos; Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M R

2015-12-01

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Platelet-derived growth factor BB and DD and angiopoietin1 are altered in follicular fluid from polycystic ovary syndrome patients.

PubMed

Scotti, Leopoldina; Parborell, Fernanda; Irusta, Griselda; De Zuñiga, Ignacio; Bisioli, Claudio; Pettorossi, Hernan; Tesone, Marta; Abramovich, Dalhia

2014-08-01

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet-derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin-5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular-endothelial cadherin, β-catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF-induced vascular permeability in the PCOS ovary by regulating endothelial-cell-junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women. © 2014 Wiley Periodicals, Inc.

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

PubMed

Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

2017-04-11

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cultures of human liver cells in simulated microgravity environment

NASA Astrophysics Data System (ADS)

Yoffe, B.; Darlington, G. J.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Khaoustov, V. I.

1999-01-01

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

PubMed Central

Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

2015-01-01

The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

A role for tight junction-associated MARVEL proteins in larval sea lamprey (Petromyzon marinus) osmoregulation.

PubMed

Kolosov, Dennis; Bui, Phuong; Donini, Andrew; Wilkie, Mike P; Kelly, Scott P

2017-10-15

This study reports on tight junction-associated MARVEL proteins of larval sea lamprey ( Petromyzon marinus ) and their potential role in ammocoete osmoregulation. Two occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln , ocln-a and tric were broadly expressed in larval lamprey, with the greatest abundance of ocln in the gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins, respectively, while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na + ] and [Cl - ] decreased, but not [Ca 2+ ], and carcass moisture content increased. In association, Ocln abundance increased in the skin and kidney, but reduced in the gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln , ocln-a and tric mRNA abundance were also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate. © 2017. Published by The Company of Biologists Ltd.

Urothelial Tight Junction Barrier Dysfunction Sensitizes Bladder Afferents

PubMed Central

Rued, Anna C.; Taiclet, Stefanie N.; Birder, Lori A.; Kullmann, F. Aura

2017-01-01

Abstract Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic voiding disorder that presents with pain in the urinary bladder and surrounding pelvic region. A growing body of evidence suggests that an increase in the permeability of the urothelium, the epithelial barrier that lines the interior of the bladder, contributes to the symptoms of IC/BPS. To examine the consequence of increased urothelial permeability on pelvic pain and afferent excitability, we overexpressed in the urothelium claudin 2 (Cldn2), a tight junction (TJ)-associated protein whose message is significantly upregulated in biopsies of IC/BPS patients. Consistent with the presence of bladder-derived pain, rats overexpressing Cldn2 showed hypersensitivity to von Frey filaments applied to the pelvic region. Overexpression of Cldn2 increased the expression of c-Fos and promoted the activation of ERK1/2 in spinal cord segments receiving bladder input, which we conceive is the result of noxious stimulation of afferent pathways. To determine whether the mechanical allodynia observed in rats with reduced urothelial barrier function results from altered afferent activity, we examined the firing of acutely isolated bladder sensory neurons. In patch-clamp recordings, about 30% of the bladder sensory neurons from rats transduced with Cldn2, but not controls transduced with GFP, displayed spontaneous activity. Furthermore, bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials from rats transduced with Cldn2 showed hyperexcitability in response to suprathreshold electrical stimulation. These findings suggest that as a result of a leaky urothelium, the diffusion of urinary solutes through the urothelial barrier sensitizes bladders afferents, promoting voiding at low filling volumes and pain. PMID:28560313

Ghrelin ameliorates intestinal barrier dysfunction in experimental colitis by inhibiting the activation of nuclear factor-kappa B

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

Cheng, Jian; Zhang, Lin; Dai, Weiqi

Aim: This study aimed to investigate the effect and underlying mechanism of ghrelin on intestinal barrier dysfunction in dextran sulfate sodium (DSS)-induced colitis. Methods and results: Acute colitis was induced in C57BL/6J mice by administering 2.5% DSS. Saline or 25, 125, 250 μg/kg ghrelin was administrated intraperitoneally (IP) to mice 1 day before colitis induction and on days 4, 5, and 6 after DSS administration. IP injection of a ghrelin receptor antagonist, [D-lys{sup 3}]-GHRP-6, was performed immediately prior to ghrelin injection. Ghrelin (125 or 250 μg/kg) could reduce the disease activity index, histological score, and myeloperoxidase activities in experimental colitis, and alsomore » prevented shortening of the colon. Ghrelin could prevent the reduction of transepithelial electrical resistance and tight junction expression, and bolstered tight junction structural integrity and regulated cytokine secretion. Ultimately, ghrelin inhibited nuclear factor kappa B (NF-κB), inhibitory κB-α, myosin light chain kinase, and phosphorylated myosin light chain 2 activation. Conclusions: Ghrelin prevented the breakdown of intestinal barrier function in DSS-induced colitis. The protective effects of ghrelin on intestinal barrier function were mediated by its receptor GHSR-1a. The inhibition of NF-κB activation might be part of the mechanism underlying the effects of ghrelin that protect against barrier dysfunction. - Highlights: • Ghrelin ameliorates intestinal barrier dysfunction in experimental colitis. • The effect of ghrelin is mediated by GHSR-1a. • Inhibition of NF-κB activation.« less

Immune Consequences of Decreasing Tumor Vasculature with Antiangiogenic Tyrosine Kinase Inhibitors in Combination with Therapeutic Vaccines

PubMed Central

Farsaci, Benedetto; Donahue, Renee N.; Coplin, Michael A.; Grenga, Italia; Lepone, Lauren M.; Molinolo, Alfredo A.; Hodge, James W.

2014-01-01

This study investigated the effects on the tumor microenvironment of combining antiangiogenic tyrosine kinase inhibitors (TKI) with therapeutic vaccines, and in particular, how vascular changes affect tumor-infiltrating immune cells. We conducted studies using a TKI (sunitinib or sorafenib) in combination with recombinant vaccines in 2 murine tumor models: colon carcinoma (MC38-CEA) and breast cancer (4T1). Tumor vasculature was measured by immunohistochemistry using 3 endothelial cell markers: CD31 (mature), CD105 (immature/proliferating), and CD11b (monocytic). We assessed oxygenation, tight junctions, compactness, and pressure within tumors, along with the frequency and phenotype of tumor-infiltrating T lymphocytes (TIL), myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM) following treatment with antiangiogenic TKIs alone, vaccine alone, or the combination of a TKI with vaccine. The combined regimen decreased tumor vasculature, compactness, tight junctions, and pressure, leading to vascular normalization and increased tumor oxygenation. This combination therapy also increased TILs, including tumor antigen-specific CD8 T cells, and elevated the expression of activation markers FAS-L, CXCL-9, CD31, and CD105 in MDSCs and TAMs, leading to reduced tumor volumes and an increase in the number of tumor-free animals. The improved antitumor activity induced by combining antiangiogenic TKIs with vaccine may be the result of activated lymphoid and myeloid cells in the tumor microenvironment, resulting from vascular normalization, decreased tumor-cell density, and the consequent improvement in vascular perfusion and oxygenation. Therapies that alter tumor architecture can thus have a dramatic impact on the effectiveness of cancer immunotherapy. PMID:25092771

Oxaliplatin-induced blood brain barrier loosening: a new point of view on chemotherapy-induced neurotoxicity

PubMed Central

Valerio Branca, Jacopo Junio; Maresca, Mario; Morucci, Gabriele; Becatti, Matteo; Paternostro, Ferdinando; Gulisano, Massimo; Ghelardini, Carla; Salvemini, Daniela

2018-01-01

Oxaliplatin is a key drug in the treatment of advanced metastatic colorectal cancer. Despite its beneficial effects in tumor reduction, the most prevalent side-effect of oxaliplatin treatment is a chemotherapy-induced neuropathy that frequently forces to discontinue the therapy. Indeed, along with direct damage to peripheral nerves, the chemotherapy-related neurotoxicity involves also the central nervous system (CNS) as demonstrated by pain chronicity and cognitive impairment (also known as chemobrain), a newly described pharmacological side effect. The presence of the blood brain barrier (BBB) is instrumental in preventing the entry of the drug into the CNS; here we tested the hypothesis that oxaliplatin might enter the endothelial cells of the BBB vessels and trigger a signaling pathway that induce the disassembly of the tight junctions, the critical components of the BBB integrity. By using a rat brain endothelial cell line (RBE4) we investigated the signaling pathway that ensued the entry of oxaliplatin within the cell. We found that the administration of 10 μM oxaliplatin for 8 and 16 h induced alterations of the tight junction (TJs) proteins zonula occludens-1 (ZO-1) and of F-actin, thus highlighting BBB alteration. Furthermore, we reported that intracellular oxaliplatin rapidly induced increased levels of reactive oxygen species and endoplasmic reticulum stress, assessed by the evaluation of glucose-regulated protein GRP78 expression levels. These events were accompanied by activation of caspase-3 that led to extracellular ATP release. These findings suggested a possible novel mechanism of action for oxaliplatin toxicity that could explain, at least in part, the chemotherapy-related central effects.

Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

PubMed Central

Oliva, Octavio M.; Karki, Suman; Surolia, Ranu; Wang, Zheng; Watson, R. Douglas; Thannickal, Victor J.; Powell, Mickie; Watts, Stephen; Kulkarni, Tejaswini; Batra, Hitesh; Bolisetty, Subhashini; Agarwal, Anupam; Antony, Veena B.

2015-01-01

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis. PMID:25835394

Identification of differentially expressed genes and pathways for intramuscular fat deposition in pectoralis major tissues of fast-and slow-growing chickens.

PubMed

Cui, Huan-Xian; Liu, Ran-Ran; Zhao, Gui-Ping; Zheng, Mai-Qing; Chen, Ji-Lan; Wen, Jie

2012-05-30

Intramuscular fat (IMF) is one of the important factors influencing meat quality, however, for chickens, the molecular regulatory mechanisms underlying this trait have not yet been determined. In this study, a systematic identification of candidate genes and new pathways related to IMF deposition in chicken breast tissue has been made using gene expression profiles of two distinct breeds: Beijing-you (BJY), a slow-growing Chinese breed possessing high meat quality and Arbor Acres (AA), a commercial fast-growing broiler line. Agilent cDNA microarray analyses were conducted to determine gene expression profiles of breast muscle sampled at different developmental stages of BJY and AA chickens. Relative to d 1 when there is no detectable IMF, breast muscle at d 21, d 42, d 90 and d 120 (only for BJY) contained 1310 differentially expressed genes (DEGs) in BJY and 1080 DEGs in AA. Of these, 34-70 DEGs related to lipid metabolism or muscle development processes were examined further in each breed based on Gene Ontology (GO) analysis. The expression of several DEGs was correlated, positively or negatively, with the changing patterns of lipid content or breast weight across the ages sampled, indicating that those genes may play key roles in these developmental processes. In addition, based on KEGG pathway analysis of DEGs in both BJY and AA chickens, it was found that in addition to pathways affecting lipid metabolism (pathways for MAPK & PPAR signaling), cell junction-related pathways (tight junction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton), which play a prominent role in maintaining the integrity of tissues, could contribute to the IMF deposition. The results of this study identified potential candidate genes associated with chicken IMF deposition and imply that IMF deposition in chicken breast muscle is regulated and mediated not only by genes and pathways related to lipid metabolism and muscle development, but also by others involved in cell junctions. These findings establish the groundwork and provide new clues for deciphering the molecular mechanisms underlying IMF deposition in poultry. Further studies at the translational and posttranslational level are now required to validate the genes and pathways identified here.

Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

PubMed

Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

2015-01-01

Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

A novel gene expression profile in lymphatics associated with tumor growth and nodal metastasis.

PubMed

Clasper, Steven; Royston, Daniel; Baban, Dilair; Cao, Yihai; Ewers, Stephan; Butz, Stefan; Vestweber, Dietmar; Jackson, David G

2008-09-15

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P

Activation of IGF-1/IGFBP-3 signaling by berberine improves intestinal mucosal barrier of rats with acute endotoxemia.

PubMed

He, Yan; Yuan, Xiaoming; Zhou, Guangrong; Feng, Aiwen

2018-01-01

Insulin-like growth factor I (IGF-I) and binding protein 3 (IGFBP-3) play a role in the maintenance of gut mucosal barrier function. Nevertheless, IGF-I/IGFBP-3 and tight junction protein (TJP) expression in small intestinal mucosa are often impaired during endotoxemia. In this model of acute endotoxemia, the regulatory effect of berberine on IGF-I/IGFBP-3 and TJP expression in ileal mucosa was evaluated. The findings revealed systemic injection of lipopolysaccharide (LPS) suppressed mRNA and protein expression of IGF-I and IGFBP-3, but berberine ameliorated their production. LPS injection inhibited occludin and claudin-1 protein generation, and this inhibitory effect of LPS was abolished by berberine. Inhibition of IGF-I/IGFBP-3 signaling by AG1024 or siRNAs reduced berberine-induced occludin and claudin-1 production. Additionally, GW9662 was found to repress berberine-induced IGF-I/IGFBP-3 expression, indicating of a cross-link between PPARγ and IGF-I/IGFBP-3 axis. Copyright © 2017 Elsevier B.V. All rights reserved.

β-1,3/1,6-Glucan alleviated intestinal mucosal barrier impairment of broiler chickens challenged with Salmonella enterica serovar Typhimurium.

PubMed

Shao, Yujing; Guo, Yuming; Wang, Zhong

2013-07-01

This study investigated the protective effect of β-1,3/1,6-glucan on gut morphology, intestinal epithelial tight junctions, and bacterial translocation of broiler chickens challenged with Salmonella enterica serovar Typhimurium. Ninety Salmonella-free Arbor Acre male broiler chickens were randomly divided into 3 groups: negative control group (NC), Salmonella Typhimurium-infected positive group (PC), and the Salmonella Typhimurium-infected group with dietary 100 mg/kg of β-1,3/1,6-glucan supplementation (T) to determine the effect of β-1,3/1,6-glucan on intestinal barrier function. Salmonella Typhimurium challenge alone significantly decreased villus height (P < 0.001), villus height/crypt depth ratio (P < 0.05), and the number of goblet cells (P < 0.001) in the jejunum at 14 d postinfection (dpi), but significantly increased the number of intestinal secretory IgA (sIgA)-expressing cells at 14 dpi (P < 0.01) and total sIgA levels in the jejunum at 7 (P < 0.05) and 14 dpi (P < 0.01) compared with the unchallenged birds (NC). Dietary β-1,3/1,6-glucan supplementation not only significantly increased villus height, villus height/crypt depth ratio, and the number of goblet cells (P < 0.01), but also increased the number of sIgA-expressing cells (P < 0.05) and sIgA content in the jejunum at 14 dpi (P < 0.01) in birds challenged with Salmonella Typhimurium in comparison with Salmonella Typhimurium challenge alone. β-1,3/1,6-Glucan addition had significant inhibitory effects (P < 0.05) on cecal Salmonella colonization levels and liver Salmonella invasion of the Salmonella Typhimurium-infected birds compared with the PC group. Intestinal tight junction proteins claudin-1, claudin-4, and occludin mRNA expression in the jejunum at 14 dpi was significantly decreased by Salmonella Typhimurium challenge alone (P < 0.01) compared with that of the NC group, whereas β-1,3/1,6-glucan supplementation significantly increased claudin-1 and occludin mRNA expression (P < 0.01) at 14 dpi in the jejunum of the Salmonella Typhimurium-infected birds in comparison with the PC group. Our results indicate that dietary β-1,3/1,6-glucan can alleviate intestinal mucosal barrier impairment in broiler chickens challenged with Salmonella Typhimurium.

Fullerene Derived Molecular Electronic Devices

NASA Technical Reports Server (NTRS)

Menon, Madhu; Srivastava, Deepak; Saini, Subbash

1998-01-01

The carbon Nanotube junctions have recently emerged as excellent candidates for use as the building blocks in the formation of nanoscale electronic devices. While the simple joint of two dissimilar tubes can be generated by the introduction of a pair of heptagon-pentagon defects in an otherwise perfect hexagonal grapheme sheet, more complex joints require other mechanisms. In this work we explore structural and electronic properties of complex 3-point junctions of carbon nanotubes using a generalized tight-binding molecular-dynamics scheme.

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

PubMed Central

Cancel, Limary M.; Tarbell, John M.

2009-01-01

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

Carbon Nanotubes: Molecular Electronic Components

NASA Technical Reports Server (NTRS)

Srivastava, Deepak; Saini, Subhash; Menon, Madhu

1997-01-01

The carbon Nanotube junctions have recently emerged as excellent candidates for use as the building blocks in the formation of nanoscale molecular electronic networks. While the simple joint of two dissimilar tubes can be generated by the introduction of a pair of heptagon-pentagon defects in an otherwise perfect hexagonal graphene sheet, more complex joints require other mechanisms. In this work we explore structural characteristics of complex 3-point junctions of carbon nanotubes using a generalized tight-binding molecular-dynamics scheme. The study of pi-electron local densities of states (LDOS) of these junctions reveal many interesting features, most prominent among them being the defect-induced states in the gap.

Spin-dependent transport in antiferromagnetic tunnel junctions

NASA Astrophysics Data System (ADS)

Merodio, P.; Kalitsov, A.; Béa, H.; Baltz, V.; Chshiev, M.

2014-09-01

We investigate the behaviour of spin transfer torque (STT) and tunnelling magnetoresistance (TMR) in epitaxial antiferromagnetic-based tunnel junctions using tight binding calculations in the framework of the Keldysh formalism. We find that the STT out-of-plane component exhibits a staggered spatial distribution similar to its in-plane component. This behaviour is specific to the use of a tunnel barrier and significantly differs from the out-of-plane torques reported in previous works using a metallic spacer. Additionally, we show that unlike conventional ferromagnetic-based tunnel junctions, the TMR can increase with applied bias and reach values comparable to typical magnetoresistances found for usual spin valves.

Chirality effect in disordered graphene ribbon junctions

NASA Astrophysics Data System (ADS)

Long, Wen

2012-05-01

We investigate the influence of edge chirality on the electronic transport in clean or disordered graphene ribbon junctions. By using the tight-binding model and the Landauer-Büttiker formalism, the junction conductance is obtained. In the clean sample, the zero-magnetic-field junction conductance is strongly chirality-dependent in both unipolar and bipolar ribbons, whereas the high-magnetic-field conductance is either chirality-independent in the unipolar or chirality-dependent in the bipolar ribbon. Furthermore, we study the disordered sample in the presence of magnetic field and find that the junction conductance is always chirality-insensitive for both unipolar and bipolar ribbons with adequate disorders. In addition, the disorder-induced conductance plateaus can exist in all chiral bipolar ribbons provided the disorder strength is moderate. These results suggest that we can neglect the effect of edge chirality in fabricating electronic devices based on the magnetotransport in a disordered graphene ribbon.

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

PubMed

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

2007-07-01

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

Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1

PubMed Central

Li, Xinbo; Olson, Carl; Lu, Shijun; Kamasawa, Naomi; Yasumura, Thomas; Rash, John E.; Nagy, James I.

2007-01-01

Among the 20 members in the connexin family of gap junction proteins, only connexin36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain-containing protein zonula occludens-1 (ZO-1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO-1. By immunofluorescence, punctate Cx36/ZO-1 colocalization was observed throughout the central nervous system of wild-type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti-Cx36 antibodies employed. By freeze-fracture replica immunogold labelling, Cx36 and ZO-1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO-1. Cx36 from mouse brain and Cx36-transfected HeLa cells was found to coimmunoprecipitate with ZO-1. Unlike other connexins that bind the second of the three PDZ domains in ZO-1, glutathione S-transferase-PDZ pull-down and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO-1, which required at most the presence of the four c-terminus amino acids of Cx36. These results demonstrating a Cx36/ZO-1 association suggest a regulatory and/or scaffolding role of ZO-1 at gap junctions that form electrical synapses between neurons in mammalian brain. PMID:15090040

Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

PubMed Central

Krishnamurthy, Shruthi; Deng, Bin; del Rio, Roxana; Buchholz, Kerry R.; Treeck, Moritz; Urban, Siniša; Boothroyd, John; Lam, Ying-Wai

2016-01-01

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T. gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. PMID:27624124

Magnetoanisotropic spin-triplet Andreev reflection in ferromagnet-Ising superconductor junctions

NASA Astrophysics Data System (ADS)

Lv, Peng; Zhou, Yan-Feng; Yang, Ning-Xuan; Sun, Qing-Feng

2018-04-01

We theoretically study the electronic transport through a ferromagnet-Ising superconductor junction. A tight-binding Hamiltonian describing the Ising superconductor is presented. Then by combining the nonequilibrium Green's function method, the expressions of Andreev reflection coefficient and conductance are obtained. A strong magnetoanisotropic spin-triplet Andreev reflection is shown, and the magnetoanisotropic period is π instead of 2 π as in the conventional magnetoanisotropic system. We demonstrate a significant increase of the spin-triplet Andreev reflection for the single-band Ising superconductor. Furthermore, the dependence of the Andreev reflection on the incident energy and incident angle are also investigated. A complete Andreev reflection can occur when the incident energy is equal to the superconducting gap, regardless of the Fermi energy (spin polarization) of the ferromagnet. For the suitable oblique incidence, the spin-triplet Andreev reflection can be strongly enhanced. In addition, the conductance spectroscopies of both zero bias and finite bias are studied, and the influence of gate voltage, exchange energy, and spin-orbit coupling on the conductance spectroscopy are discussed in detail. The conductance exhibits a strong magnetoanisotropy with period π as the Andreev reflection coefficient. When the magnetization direction is parallel to the junction plane, a large conductance peak always emerges at the superconducting gap. This work offers a comprehensive and systematic study of the spin-triplet Andreev reflection and has an underlying application of π -periodic spin valve in spintronics.

Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport

PubMed Central

Herrmann, Jeremy R.

2016-01-01

In the mid-20th century, Hans Ussing developed a chamber that allowed for the simultaneous measurement of current and labeled probe flux across epithelia. Using frog skin as a model, Ussing used his results to propose mechanisms of transcellular Na+ and K+ transport across apical (exterior/luminal) and basolateral (interior) membranes that is essentially unchanged today. Others took advantage of Ussing's chambers to study mucosal tissues, including bladder and intestines. It quickly became clear that, in some tissues, passive paracellular flux, i.e., across the tight junction, was an important component of overall transepithelial transport. Subsequent work demonstrated that activation of the apical Na+-glucose cotransporter SGLT1 regulated paracellular permeability such that intestinal paracellular transport could coordinate with and amplify transcellular transport. Intermediates in this process include activation of p38 MAPK, the apical Na+/H+ exchanger NHE3, and myosin light chain kinase (MLCK). Investigators then focused on these processes in disease. They found that TNF induces barrier dysfunction via MLCK activation and downstream caveolin-1-dependent endocytosis of the tight junction protein occludin. TNF also inhibited NHE3, and both barrier loss and PKCα-dependent NHE3 inhibition were required for TNF-induced acute diarrhea, emphasizing the interplay between transcellular and paracellular transport. Finally, studies using immune-mediated inflammatory bowel disease models showed that mice lacking epithelial MLCK were initially protected, but became ill as epithelial damage progressed and provided a tight junction-independent means of barrier loss. None of these advances would have been possible without the insights provided by Ussing and others using Ussing's ingenious, and still useful, chambers. PMID:26702131

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

PubMed Central

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

2013-01-01

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

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

PubMed Central

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

2015-01-01

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

Colon dysregulation in methamphetamine self-administering HIV-1 transgenic rats

PubMed Central

Bradaric, Brinda D.; Dodiya, Hemraj B.; Ohene-Nyako, Michael; Forsyth, Christopher B.; Keshavarzian, Ali; Shaikh, Maliha; Napier, T. Celeste

2018-01-01

The integrity and function of the gut is impaired in HIV-infected individuals, and gut pathogenesis may play a role in several HIV-associated disorders. Methamphetamine is a popular illicit drug abused by HIV-infected individuals. However, the effect of methamphetamine on the gut and its potential to exacerbate HIV-associated gut pathology is not known. To shed light on this scenario, we evaluated colon barrier pathology in a rat model of the human comorbid condition. Intestinal barrier integrity and permeability were assessed in drug-naïve Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats, and in Tg and non-Tg rats instrumented with jugular cannulae trained to self-administer methamphetamine or serving as saline-yoked controls. Intestinal permeability was determined by measuring the urine content of orally gavaged sugars. Intestinal barrier integrity was evaluated by immunoblotting or immunofluorescence of colon claudin-1 and zonula occludens-1 (ZO-1), two major tight junction proteins that regulate gut epithelial paracellular permeability. Both non-Tg and Tg rats self-administered moderate amounts of methamphetamine. These amounts were sufficient to increase colon permeability, reduce protein level of claudin-1, and reduce claudin-1 and ZO-1 immunofluorescence in Tg rats relative to non-Tg rats. Methamphetamine decreased tight junction immunofluorescence in non-Tg rats, with a similar, but non-significant trend observed in Tg rats. However, the effect of methamphetamine on tight junction proteins was subthreshold to gut leakiness. These findings reveal that both HIV-1 proteins and methamphetamine alter colon barrier integrity, and indicate that the gut may be a pathogenic site for these insults. PMID:29293553

Colon dysregulation in methamphetamine self-administering HIV-1 transgenic rats.

PubMed

Persons, Amanda L; Bradaric, Brinda D; Dodiya, Hemraj B; Ohene-Nyako, Michael; Forsyth, Christopher B; Keshavarzian, Ali; Shaikh, Maliha; Napier, T Celeste

2018-01-01

The integrity and function of the gut is impaired in HIV-infected individuals, and gut pathogenesis may play a role in several HIV-associated disorders. Methamphetamine is a popular illicit drug abused by HIV-infected individuals. However, the effect of methamphetamine on the gut and its potential to exacerbate HIV-associated gut pathology is not known. To shed light on this scenario, we evaluated colon barrier pathology in a rat model of the human comorbid condition. Intestinal barrier integrity and permeability were assessed in drug-naïve Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats, and in Tg and non-Tg rats instrumented with jugular cannulae trained to self-administer methamphetamine or serving as saline-yoked controls. Intestinal permeability was determined by measuring the urine content of orally gavaged sugars. Intestinal barrier integrity was evaluated by immunoblotting or immunofluorescence of colon claudin-1 and zonula occludens-1 (ZO-1), two major tight junction proteins that regulate gut epithelial paracellular permeability. Both non-Tg and Tg rats self-administered moderate amounts of methamphetamine. These amounts were sufficient to increase colon permeability, reduce protein level of claudin-1, and reduce claudin-1 and ZO-1 immunofluorescence in Tg rats relative to non-Tg rats. Methamphetamine decreased tight junction immunofluorescence in non-Tg rats, with a similar, but non-significant trend observed in Tg rats. However, the effect of methamphetamine on tight junction proteins was subthreshold to gut leakiness. These findings reveal that both HIV-1 proteins and methamphetamine alter colon barrier integrity, and indicate that the gut may be a pathogenic site for these insults.

Acute and Chronic Deficits in the Urinary Bladder after Spinal Contusion Injury in the Adult Rat

PubMed Central

Herrera, Juan J.; Haywood-Watson, Ricky J.L.

2010-01-01

Abstract Traumatic spinal cord injury (SCI) permanently alters bladder function in humans. Hematuria and cystitis occur in both human SCI as well as in rodent models of SCI. Others have reported early SCI-dependent disruption to bladder uroepithelial integrity that results in increased permeability to urine and urine-borne substances. This can result in cystitis, or inflammation of the bladder, an ongoing pathological condition present throughout the chronic phase of SCI in humans. The goals of our study were twofold: (1) to begin to examine the inflammatory and molecular changes that occur within the bladder uroepithelium using a clinically-relevant spinal contusion model of injury, and (2) to assess whether these alterations continue into the chronic phase of SCI. Rats received either moderate SCI or sham surgery. Urine was collected from SCI and sham subjects over 7 days or at 7 months to assess levels of excreted proteins. Inflammation in the bladder wall was assessed via biochemical and immunohistochemical methods. Bladder tight junction proteins, mediators of uroepithelial integrity, were also measured in both the acute and chronic phases of SCI. Urine protein and hemoglobin levels rapidly increase following SCI. An SCI-dependent elevation in numbers of neutrophils within the bladder wall peaked at 48 h. Bladder tight junction proteins demonstrate a rapid but transient decrease as early as 2 h post-SCI. Surprisingly, elevated levels of urine proteins and significant deficits in bladder tight junction proteins could be detected in chronic SCI, suggesting that early pathological changes to the bladder may continue throughout the chronic phase of injury. PMID:19891526

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

PubMed

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

2018-06-07

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

Morphofunctional aspects of the blood-brain barrier.

PubMed

Nico, Beatrice; Ribatti, Domenico

2012-01-01

The blood-brain barrier (BBB) selectively controls the homeostasis of the Central Nervous System (CNS) environment by the specific structural and biochemical features of the endothelial cells, pericytes and glial endfeet, which represent the cellular components of the mature BBB. Endothelial tight junctions (TJs) are the most important structural component of the BBB, and molecular alteration in the phosphorylation state of some TJs proteins, like ZO-1 or occludin, are crucial in determining alterations in the control of BBB vascular permeability. Astrocytes endfeet enveloping the vessels wall, are considered important in the induction and maintenance of the BBB, through secretion of soluble factors, which modulate the expression of enzymatic complexes and antigens by endothelial cells and TJs - associated proteins. Moreover, astrocytes control water flux at BBB site by expressing a specific water channel, namely aquaporin-4 (AQP4), involved in the molecular composition of the orthogonal particles arrays (OAPs) on the perivascular glial endfeet and tightly coupled with the maintenance of the BBB integrity. Disruption of the BBB is a consistent event occurring in the development of several CNS diseases, including demyelinating lesions in the course of relapsing multiple sclerosis, stroke, Duchenne muscular dystrophy (DMD), but also mechanical injures, neurological insults, septic encephalopathy, brain tumors, permanent ischemia or transient ischemia followed by reperfusion. In most cases, these pathological conditions are associated with an increase in microvascular permeability, vasogenic edema, swollen atrocyte endfeet, and BBB disruption.

Claudins and renal salt transport.

PubMed

Muto, Shigeaki; Furuse, Mikio; Kusano, Eiji

2012-02-01

Tight junctions (TJs) are the most apical component of junctional complexes and regulate the movement of electrolytes and solutes by the paracellular pathway across epithelia. The defining ultrastructural features of TJs are strands of transmembrane protein particles that adhere to similar strands on adjacent cells. These strands are mainly composed of linearly polymerized integral membrane proteins called claudins. Claudins comprise a multigene family consisting of more than 20 members in mammals. Recent work has shown that claudins form barriers, determined by the paracellular electrical resistance and charge selectivity, and pores in the TJ strands. The paracellular pathways in renal tubular epithelia such as the proximal tubule, which reabsorbs the largest fraction of filtered NaCl and water, are important routes for the transport of electrolytes and water. Their transport characteristics vary among different nephron segments. Multiple claudins are expressed at TJs of individual nephron segments in a nephron segment-specific manner. Among them, claudin-2 is highly expressed at TJs of proximal tubules, which are leaky epithelia. Overexpression and knockdown of claudin-2 in epithelial cell lines, and knockout of the claudin-2 gene in mice, have demonstrated that claudin-2 forms high-conductance cation-selective pores in the proximal tubule. Here, we review the renal physiology of paracellular transport and the physiological roles of claudins in kidney function, especially claudin-2 and proximal tubule paracellular NaCl transport.

Nance-Horan syndrome protein, NHS, associates with epithelial cell junctions.

PubMed

Sharma, Shiwani; Ang, Sharyn L; Shaw, Marie; Mackey, David A; Gécz, Jozef; McAvoy, John W; Craig, Jamie E

2006-06-15

Nance-Horan syndrome, characterized by congenital cataracts, craniofacial, dental abnormalities and mental disturbances, is an X-linked disorder with significant phenotypic heterogeneity. Affected individuals have mutations in the NHS (Nance-Horan syndrome) gene typically resulting in premature truncation of the protein. This report underlines the complexity of the regulation of the NHS gene that transcribes several isoforms. We demonstrate the differential expression of the two NHS isoforms, NHS-A and NHS-1A, and differences in the subcellular localization of the proteins encoded by these isoforms. This may in part explain the pleiotropic features of the syndrome. We show that the endogenous and exogenous NHS-A isoform localizes to the cell membrane of mammalian cells in a cell-type-dependent manner and that it co-localizes with the tight junction (TJ) protein ZO-1 in the apical aspect of cell membrane in epithelial cells. We also show that the NHS-1A isoform is a cytoplasmic protein. In the developing mammalian lens, we found continuous expression of NHS that became restricted to the lens epithelium in pre- and postnatal lens. Consistent with the in vitro findings, the NHS-A isoform associates with the apical cell membrane in the lens epithelium. This study suggests that disturbances in intercellular contacts underlie cataractogenesis in the Nance-Horan syndrome. NHS is the first gene localized at TJs that has been implicated in congenital cataracts.

Claudins 1, 2, 3, 4, 5 and 7 in solar keratosis and squamocellular carcinoma of the skin

PubMed Central

Hintsala, Hanna-Riikka; Siponen, Maria; Haapasaari, Kirsi-Maria; Karihtala, Peeter; Soini, Ylermi

2013-01-01

Claudins are tight junction proteins regulating the paracellular permeability of cell layers. We investigated the expression of claudins 1, 2, 3, 4, 5 and 7 in a sample set consisting of a total of 93 cases representing normal skin, actinic keratoses and squamous cell carcinomas of the skin. There were several changes found in claudin expression. Claudin 1 appeared to be progressively decreased in solar keratosis and skin squamous cell carcinomas compared to normal skin while expression of claudin 2 was increased. With claudins 3 and 5 occasional immunoreactivity was found in squamous cell carcinomas. Claudins 4 and 7 were variably expressed in skin neoplasia compared to normal skin. According to the results expression of claudins 1 and 2 change in parallel with the severity of the epidermal preneoplastic and neoplastic lesions thus probably influencing the disturbed epithelial polarity characteristic of these lesions. Claudin 1 under- and claudin 2 overexpression also lead to a leakier epithelial barrier function of the skin with a resulting damage to skin epithelial resistance. Other claudins investigated in this study did not show progressive changes even though occasional overexpression of them was found in skin squamous cell carcinoma. PMID:24294371

ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

PubMed

Zhang, Hu; Zhang, Shuhong; Zhang, Jilin; Liu, Dongxin; Wei, Jiayi; Fang, Wengang; Zhao, Weidong; Chen, Yuhua; Shang, Deshu

2018-05-01

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

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

PubMed

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

2016-12-01

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

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

PubMed

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

2007-09-01

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

Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

PubMed Central

Gungor-Ordueri, N. Ece; Celik-Ozenci, Ciler

2014-01-01

In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326

Glucocorticoid Induction of Occludin Expression and Endothelial Barrier Requires Transcription Factor p54 NONO

PubMed Central

Keil, Jason M.; Liu, Xuwen; Antonetti, David A.

2013-01-01

Purpose. Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties. Methods. OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system. Results. MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment. Conclusions. p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema. PMID:23640037

Glucocorticoid induction of occludin expression and endothelial barrier requires transcription factor p54 NONO.

PubMed

Keil, Jason M; Liu, Xuwen; Antonetti, David A

2013-06-12

Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties. OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system. MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment. p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium

PubMed Central

Faris, Andrea N.; Ganesan, Shyamala; Chattoraj, Asamanja; Chattoraj, Sangbrita S.; Comstock, Adam T.; Unger, Benjamin L.; Hershenson, Marc B.

2016-01-01

Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia. PMID:27119973

Transcriptome analysis reveals regional and temporal differences in mucosal immune system development in the small intestine of neonatal calves.

PubMed

Liang, Guanxiang; Malmuthuge, Nilusha; Bao, Hua; Stothard, Paul; Griebel, Philip J; Guan, Le Luo

2016-08-11

Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum (n = 3) and at 7 (n = 5), 21 (n = 5), and 42 (n = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Nutrient sensing signaling integrates nutrient metabolism and intestinal immunity in grass carp, Ctenopharyngodon idellus after prolonged starvation.

PubMed

Li, Shan; Li, Jiabo; Zhao, Yongliang; Zhang, Qin; Wang, Qingchao

2017-12-01

Starvation has been shown to affect growth and nutrient metabolism in fish; however, little information about the nutrient sensing signaling and mucosal adaptive immunity in fish was known. In the present study, grass carp was starved for 8weeks to simulate the natural aquaculture practice in Hubei during winter. The histology of liver was significantly affected with decreased expression of tight junction proteins including claudin-3, claudin-b and ZO-1. Muscle gene expression was also affected, with decreased expression of muscle growth promoting factors such as Myogenin, MyoD, Myf5, and increased expression of muscle degradation factors, such as CathepsinD. In addition, mucosal adaptive immunity was also significantly affected, with decreased expression of antibodies including IgZ and IgM in gut. Along with these changes was the inhibition of several nutrient sensing signaling including MAPK and TOR signaling, which leads to the inhibition of the synthesis of protein including immunoglobulin. The increased phosphorylation of eIF2α not only inhibited the translation, but also resulted in the decreased expression of IkB and increased expression of NF-B, with the activation of pro-inflammatory genes including IL8 and TNF. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transcriptional Modulation of Genes Encoding Structural Characteristics of Differentiating Enterocytes During Development of a Polarized Epithelium In Vitro

PubMed Central

Halbleib, Jennifer M.; Sääf, Annika M.

2007-01-01

Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell–cell adhesion–initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes. PMID:17699590

Perineurial-like Cells and EMA Expression in the Suprachoroidal Region of the Human Eye.

PubMed

Gilbert, Andrea R; Chévez-Barrios, Patricia; Cykowski, Matthew D

2018-05-01

The suprachoroidal region of the eye comprises vascular channels, melanocytes, and thin fibroblasts with elongated cytoplasm that are positioned directly adjacent to the densely collagenous sclera. Morphological similarities between these suprachoroidal fibroblasts and arachnoid cells and perineurial cells have been recognized, but whether these fibroblasts have a perineurial cell-like immunophenotype is not known. To further examine the relationship of these three cell types, we investigated the comparative expression of epithelial membrane antigen (EMA), the tight junction protein claudin-1, glucose transporter-1 (Glut-1), and CD34 in suprachoroidal fibroblasts, arachnoid of the optic nerve sheath, and perineurium of ciliary nerves in eight human eye specimens. Granular, diffuse, and cytoplasmic EMA expression was seen in suprachoroidal fibroblasts, but this was not contiguous with the similar pattern of EMA expression in adjacent perineurium and arachnoid. CD34 expression in suprachoroidal fibroblasts was also seen, similar to arachnoid and perineurium. Claudin-1 and Glut-1 were not consistently expressed in suprachoroidal fibroblasts, distinguishing them from perineurial cells in particular and suggesting that these fibroblasts do not arise directly from adjacent arachnoid or perineurium. Nonetheless, the overlapping morphology and protein expression suggest phenotypic similarities in these cells that protect and support adjacent retina, optic nerve, and peripheral nerve.

Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability.

PubMed

Cani, P D; Possemiers, S; Van de Wiele, T; Guiot, Y; Everard, A; Rottier, O; Geurts, L; Naslain, D; Neyrinck, A; Lambert, D M; Muccioli, G G; Delzenne, N M

2009-08-01

Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes.

Nanotopographical Cues for Modulating Fibrosis and Drug Delivery

NASA Astrophysics Data System (ADS)

Walsh, Laura Aiko Michelle

Nanotopography in the cellular microenvironment provides biological cues and therefore has potential to be a useful tool for directing cellular behavior. Fibrotic encapsulation of implanted devices and materials can wall off and eventually cause functional failure of the implant. Drug delivery requires penetrating the epithelium, which encapsulates the body and provides a barrier to separate the body from its external environment. Both of these challenges could be elegantly surmounted using nanotopography, which would harness innate cellular responses to topographic cues to elicit desired cellular behavior. To this end, we fabricated high and low aspect ratio nanotopographically patterned thin films. Using scanning electron microscopy, real time polymerase chain reaction, immunofluorescence microscopy, in vitro drug delivery assays, transmission electron microscopy, inhibitor studies, and rabbit and rat in vivo drug delivery studies, we investigated cellular response to our nanotopographic thin films. We determined that high aspect ratio topography altered fibroblast morphology and decreased proliferation, possibly due to decreased protein adsorption. The fibroblasts also down regulated expression of mRNA of key factors associated with fibrosis, such as collagens 1 and 3. Low aspect ratio nanotopography increased drug delivery in vitro across an intestinal epithelial model monolayer by increasing paracellular permeability and remodeling the tight junction. This increase in drug delivery required integrin engagement and MLCK activity, and is consistent with the increased focal adhesion formation. Tight junction remodeling was also observed in a multilayered keratinocyte model, showing this mechanism can be generalized to multiple epithelium types. By facilitating direct contact of nanotopography with the viable epidermis using microneedles to pierce the stratum corneum, we are able to transdermally deliver a 150 kiloDalton, IgG-based therapeutic in vivo..

Soyabean glycinin depresses intestinal growth and function in juvenile Jian carp (Cyprinus carpio var Jian): protective effects of glutamine.

PubMed

Jiang, Wei-Dan; Hu, Kai; Zhang, Jin-Xiu; Liu, Yang; Jiang, Jun; Wu, Pei; Zhao, Juan; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

2015-11-28

This study investigated the effects of glycinin on the growth, intestinal oxidative status, tight junction components, cytokines and apoptosis signalling factors of fish. The results showed that an 80 g/kg diet of glycinin exposure for 42 d caused poor growth performance and depressed intestinal growth and function of juvenile Jian carp (Cyprinus carpio var. Jian). Meanwhile, dietary glycinin exposure induced increases in lipid peroxidation and protein oxidation; it caused reductions in superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities; and it increased MnSOD, CuZnSOD, GPx1b and GPx4a mRNA levels, suggesting an adaptive mechanism against stress in the intestines of fish. However, dietary glycinin exposure decreased both the activity and mRNA levels of nine isoforms of glutathione-S-transferase (GST) (α, μ, π, ρ, θ, κ, mGST1, mGST2 and mGST3), indicating toxicity to this enzyme activity and corresponding isoform gene expressions. In addition, glycinin exposure caused partial disruption of intestinal cell-cell tight junction components, disturbances of cytokines and induced apoptosis signalling in the distal intestines>mid intestines>proximal intestines of fish. Glycinin exposure also disturbed the mRNA levels of intestinal-related signalling factors Nrf2, Keap1a, Keap1b, eleven isoforms of protein kinase C and target of rapamycin/4E-BP. Interestingly, glutamine was observed to partially block those negative influences. In conclusion, this study indicates that dietary glycinin exposure causes intestinal oxidative damage and disruption of intestinal physical barriers and functions and reduces fish growth, but glutamine can reverse those negative effects in fish. This study provides some information on the mechanism of glycinin-induced negative effects.

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

PubMed Central

Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

2013-01-01

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408