Composition Of Tight Junctions Research Articles

Plant-derived triterpene celastrol ameliorates oxygen glucose deprivation-induced disruption of endothelial barrier assembly via inducing tight junction proteins

BackgroundThe integrity and functions of blood-brain barrier (BBB) are regulated by the expression and organization of tight junction proteins. ObjectiveThe present study was designed to explore whether plant-derived triterpenoid celastrol could regulate tight junction integrity in murine brain endothelial bEnd3 cells. MethodsWe disrupted the tight junctions between endothelial bEnd3 cells by oxygen glucose deprivation (OGD). We investigated the effects of celastrol on the permeability of endothelial monolayers by measuring transepithelial electrical resistance (TEER). To clarify the tight junction composition, we analyzed the expression of tight junction proteins by RT-PCR and Western blotting techniques. ResultsWe found that celastrol recovered OGD-induced TEER loss in a concentration-dependent manner. Celastrol induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells. As a result, celastrol effectively maintained tight junction integrity and inhibited macrophage migration through endothelial monolayers against OGD challenge. Further mechanistic studies revealed that celastrol induced the expression of occludin and ZO-1) via activating MAPKs and PI3K/Akt/mTOR pathway. We also observed that celastrol regulated claudin-5 expression through different mechanisms. ConclusionThe present study demonstrated that celastrol effectively protected tight junction integrity against OGD-induced damage. Thus, celastrol could be a drug candidate for the treatment of BBB dysfunction in various diseases.

Loss of PTPN2 in Intestinal Epithelial Cells Upregulates a Novel STAT‐Dependent Mechanism of Claudin‐2 Expression

Inflammatory bowel disease (IBD) is a multi‐factorial condition characterized by elevated levels of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Genome‐wide‐association‐studies (GWAS) revealed that individuals with single nucleotide polymorphic mutations in the locus of the protein tyrosine phosphatase non‐receptor type 2 (PTPN2) gene that encodes T‐cell protein tyrosine phosphatase (TCPTP), have an increased susceptibility to IBD. We have reported that reduced TCPTP expression increased effects of IFN‐γ on Signal Transducer and Activator of Transcription (STAT) signaling and led to a greater reduction in barrier function in intestinal epithelial cells (IEC). The protein composition of tight junctions (TJ) determines the tightness of the barrier. Claudin‐2 is a TJ protein that is upregulated in IBD and increases paracellular permeability to cations thus reducing transepithelial electrical resistance (TER). We have previously shown that transient knockdown of TCPTP permits increased expression of claudin‐2 through Janus Kinase (JAK)‐STAT signaling. The aim of the present study was to identify the molecular mechanisms underlying STAT regulation of claudin‐2 expression in PTPN2 (TCPTP)‐knockdown (PTPN2‐KD) HT‐29 IEC. Western blot and immunofluorescence studies revealed that basal STAT1 phosphorylation (Y701) and claudin‐2 expression were increased in unchallenged PTPN2‐KD cells compared to control shRNA transfected cells, and this effect was greatly enhanced by IFN‐γ treatment. Protein analysis of soluble and insoluble fractions showed increased claudin‐2 in the insoluble fraction in KD cells compared to control cells. siRNA silencing of STAT1 mRNA levels significantly decreased claudin‐2 protein and mRNA expression. To confirm STAT binding to the claudin‐2 promoter region, we used Chromatin immunoprecipitation (ChIP) assay to immunoprecipitate STAT1 from IFN‐γ‐treated PTPN2‐KD and control IEC with anti‐STAT1 antibody, and probed the associated DNA with primers against the claudin‐2 promoter. ChIP analysis showed increased STAT1 binding to the claudin‐2 promoter region in KD cells compared to control cells. Next we determined if STAT1 activates claudin‐2 promoter activity through the STAT binding site. We performed a luciferase reporter assay using a human claudin‐2 promoter‐luciferase construct expressed in PTPN2‐KD or Con‐shRNA IEC. Luciferase activity was significantly increased (214 ± 9%, p<0.0001; n=3) in PTPN2‐KD cells over control IEC. In further studies using two different constructs of the claudin‐2 promoter region with mutations of the STAT binding site, there was a significant decrease in promoter activity (43±15%, p<0.0001; n=3) when compared to full length claudin‐2 promoter activity in KD cells, but no significant difference with control cells. In summary, we have demonstrated that STAT1 regulates claudin‐2 expression through binding to its promoter region under conditions of decreased activity of the IBD candidate gene, PTPN2.

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells.

A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus.

Hereditary barrier-related diseases involving the tight junction: lessons from skin and intestine

The tight junction (TJ) regulates paracellular barrier properties. TJs are composed of transmembrane proteins, i.e., claudins, occludin, tricellulin and junctional adhesion molecules as well as TJ plaque proteins. Their relative abundance and composition determines epithelial tightness. TJs undergo rapid regulation by various signalling pathways, either directly addressing TJ transmembrane proteins or via plaque proteins and the cytoskeleton. In the skin, TJs exert predominantly barrier functions, while in the intestine they also mediate paracellular ion and water transport. In diseases, TJs can either be primarily affected (hereditary TJ defects) or changes can result from secondary regulatory inputs as, e.g., in inflammatory diseases (secondary TJ defects). Secondary TJ defects can maintain disease activity, e.g., by enhanced antigen leak. This review discusses TJ composition, function and regulation as well as primary and secondary tight junction defects in a comparative manner in skin and intestine in order to elucidate similarities and differences.

Endothelial-Leukocyte Interaction in Severe Malaria: Beyond the Brain.

Malaria is the most important parasitic disease worldwide, accounting for 1 million deaths each year. Severe malaria is a systemic illness characterized by dysfunction of brain tissue and of one or more peripheral organs as lungs and kidney. The most severe and most studied form of malaria is associated with cerebral complications due to capillary congestion and the adhesion of infected erythrocytes, platelets, and leukocytes to brain vasculature. Thus, leukocyte rolling and adhesion in the brain vascular bed during severe malaria is singular and distinct from other models of inflammation. The leukocyte/endothelium interaction and neutrophil accumulation are also observed in the lungs. However, lung interactions differ from brain interactions, likely due to differences in the blood-brain barrier and blood-air barrier tight junction composition of the brain and lung endothelium. Here, we review the importance of endothelial dysfunction and the mechanism of leukocyte/endothelium interaction during severe malaria. Furthermore, we hypothesize a possible use of adjunctive therapies to antimalarial drugs that target the interaction between the leukocytes and the endothelium.

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

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

570 In Vivo PTPN2-Deficiency and a Dominant-Negative PTPN2 Mutation Cause Increased Intestinal Permeability and Alter Tight Junction Composition

We have previously generated C57BL/6J-Tg(Car1-cre)5Flt transgenic mice (CAC) with large intestine, epithelial cell-specific expression of Cre recombinase and demonstrated that they can be used to study colorectal cancer. Here we report a novel observation with CAC mice that expands their utility as an experimental model for colitis and inflammation-induced colon cancer research. CAC mice were crossed to ROSA26R reporter mice (ROSA) that have a lox-STOP-lox site controlling β-galactosidase (β-gal) expression. In study 1, 8-wk-old CAC;ROSA mice were treated with 0, 0.65, 1.35, or 2% DSS in drinking water for 5 d. Proximal (CoP) and distal colon (CoD) segments were harvest 10 d after ending DSS treatment. The colon was fixed in 10% neutral buffered formalin, paraffin embedded, and processed for determination of an epithelial damage score (reflecting % surface ulceration and% regenerating epithelium) and for immunohistochemical detection of β-gal (an indicator of Cre-mediated recombination). In control mice (0% DSS) β-gal staining was superficial in CoP while 3% of CoD epithelium had β-gal staining from crypt base to luminal surface. DSS treatment caused a dose-dependent increase in damage score for the CoP (0% DSS = 0, 0.65%DSS = 30.5±4.3, 1.35% DSS = 53.6±4.1, 2% DSS = 58.5±4.9%) and CoD epithelium (0, 24.7±8.0, 46.7±9.8, 48.4±10.6%) and this was associated with increased β-gal staining (CoP = 0.7±0.2, 20.4±1.8, 41.0±2.9, 45.9±4.6%; CoD = 3.0±0.54, 20.4±5.2, 35.4±6.9, 42.2±9.4%). Interestingly, 60% regenerating epithelium was β-gal positive regardless of the DSS dose or the colon segment examined; this new staining was seen from crypt base to luminal surface in both CoP and CoD. In study 2, we examined whether β-gal induction persisted after the epithelium had healed, reflecting Cre mediated recombination in intestinal stem cells. 8-wk-old CAC;ROSA mice were treated with 1.35% DSS and β-gal staining was assessed in colon at 10 and 30 d post DSS treatment. Epithelial β-gal staining was low in the control (CoP = 0.8±0.2%; CoD = 6.6±0.8%) and increased with the epithelial damage score at 10 d (CoP damage = 18.7±3.4%, β-gal = 17.4±2.9%; CoD damage = 27.6±7.3%, β-gal = 33.6±6.8%). At 30 d, all ulcers had healed and the % regenerating epithelium was reduced by >45% in each segment compared to the 10 d timepoint. However, epithelial βgal staining remained high in both colon segments (CoP = 18.9±1.8%; CoD = 44.4±5.2%), especially in the epithelium of the healed ulcer (CoP=75.2±4.4%; CoD = 89.4±2.2%). These studies show that in CAC mice, DSS-mediated epithelial damage can be used to induce a persistent, Cre-mediated recombination of floxed alleles. This will permit examination of the function of genes in colon epithelium during experimental colitis and inflammationinduced colon cancer.

Knockout Mice Reveal Key Roles for Claudin 18 in Alveolar Barrier Properties and Fluid Homeostasis

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Zinc Supplementation Modifies Tight Junctions and Alters Barrier Function of CACO-2 Human Intestinal Epithelial Layers

Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function. The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells. Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7. Fifty- and 100-μM zinc concentrations (control medium is 2 μM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation. Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of "nutraceutical" substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference-mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical-basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.

BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma

The acquisition of a mesenchymal phenotype is a critical step in the metastatic progression of epithelial carcinomas. Adherens junctions (AJs) are required for suppressing this epithelial-mesenchymal transition (EMT) but less is known about the role of tight junctions (TJs) in this process. Here, we investigated the functions of blood vessel epicardial substance (BVES, also known as POPDC1 and POP1), an integral membrane protein that regulates TJ formation. BVES was found to be underexpressed in all stages of human colorectal carcinoma (CRC) and in adenomatous polyps, indicating its suppression occurs early in transformation. Similarly, the majority of CRC cell lines tested exhibited decreased BVES expression and promoter DNA hypermethylation, a modification associated with transcriptional silencing. Treatment with a DNA-demethylating agent restored BVES expression in CRC cell lines, indicating that methylation represses BVES expression. Reexpression of BVES in CRC cell lines promoted an epithelial phenotype, featuring decreased proliferation, migration, invasion, and anchorage-independent growth; impaired growth of an orthotopic xenograft; and blocked metastasis. Conversely, interfering with BVES function by expressing a dominant-negative mutant in human corneal epithelial cells induced mesenchymal features. These biological outcomes were associated with changes in AJ and TJ composition and related signaling. Therefore, BVES prevents EMT, and its epigenetic silencing may be an important step in promoting EMT programs during colon carcinogenesis.

Dietary methionine restriction enhances tight junction barrier functions in rat colon but not rat ileum

Dietary methionine restriction (MR) (an 80% reduction of methionine level) has been shown previously to improve tight junctional barrier function in both LLC-PK1 renal epithelial cells (Skrovanek et al., 2008) and rat colonic epithelium (Ramalingam et al., 2010). We investigated to see if this phenomenon also holds in a structurally and functionally distinct gastrointestinal tissue, rat ileum. Rat plasma methionine level started to decrease as early as 1 week on experimental diet and reached a constant level (~20% decrease) from 2 weeks onwards up to 9 weeks. Despite the change in plasma methionine level, we were not able to detect any differences in ileal transepithelial resistance, or paracellular mannitol as well as lactulose transepithelial fluxes between the control and MR groups. Western blot analysis of ileal mucosal scrapes revealed no statistically significant changes in protein abundance of the tight junctional proteins occludin, claudin-1, 2, 3, 4, 5, or 7. Although a prominent improvement was observed on barrier function (and tight junction composition) in rat distal colon following MR, no significant epithelial tight junctional changes were detected in rat distal ileum. Our data suggest that MR may have different effects on TJ composition and barrier function depending on epithelial tissue location and function.

Identifying downstream mediators linking JAM‐A to Barrier Function

Intestinal barrier function is regulated by epithelial tight junctions (TJs), allowing for absorption of nutrients and exclusion of unwanted substances. A TJ-associated transmembrane protein shown to play a role in regulation of barrier function is Junctional Adhesion Molecule-A (JAM-A). JAM-A has a short cytoplasmic tail that associates with scaffold proteins and two extracellular immunoglobulin-like domains that allow for its homodimerization on the surface of the same cell. Despite several structural and functional studies, mechanistic details linking JAM-A to TJ regulation is lacking. We have previously shown that JAM-A dimerization affects epithelial cell migration through close apposition of PDZ-GEF2 and Afadin resulting in activation of the small GTPase Rap1a. We now report that knockdown of Afadin and PDZ-GEF1 and 2, but not Rap1a or Rap1b, impair barrier function in vitro. Given that loss of Rap1 did not affect barrier, we assessed the role of other GTPases activated by PDZ-GEFs and determined that knockdown of Rap2 disrupts barrier in a manner similar to that observed with loss of JAM-A. In addition, our preliminary studies suggest that JAM-A dimerization differentially regulates expression of barrier-forming claudin proteins. These findings suggest that JAM-A clusters Afadin and PDZ-GEFs to activate Rap2 and regulate barrier by altering the protein composition of TJs(Supported by NIH DK061379)

The Biology of Epithelial Cell Tight Junctions in the Kidney

Nearly 50 years have lapsed since the tight junction between epithelial cells was first identified by electron microscopy. The tight junction was once viewed as a static structure providing a barrier to paracellular movement and restricting proteins to the apical or basolateral membrane. Recent insights into the molecular composition of tight junctions reveal surprising complexity and dynamic regulation. Epithelia along the nephron exemplify a diversity of tight junctions that contribute to more than a 100-fold difference in permeability from the proximal tubule to the collecting duct. Tight junctions along the nephron form during kidney development and must reassemble after tubular injury. Hereditary diseases, animal models, and cell culture studies provide a variety of new perspectives on the function of tight junctions in health and disease.

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

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

Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10(6) CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.

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

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

Wirkung von VEGF165und dem VEGF-Aptamer Pegaptanib (Macugen®) auf die Zusammensetzung der Tight Junctions mikrovaskulärer Endothelzellen aus der Retina

VEGF signalling is deregulated in diabetic retinopathy. Therefore, VEGF inhibitors like the modified RNA-oligonucleotide pegaptanib (VEGF aptamer, Macugen) which inhibits the interaction of VEGF(165) with its receptors, are currently being discussed as therapeutic options in the treatment of diabetic retinopathy. VEGF(165) does not only stimulate the proliferation and migration of endothelial cells but also induces delocalization of occludin which is part of the so-called tight junctions of endothelial cells likely associated with the breakdown of the blood-retina barrier. To further investigate the mechanisms of action of VEGF and its inhibitor, we studied the influence of VEGF(165) and/or pegaptanib on the protein composition of tight junctions in immortalised endothelial cells of the bovine retina (iBREC) by immunofluorescence staining. The tight junction proteins ZO-1, occludin and claudin-5 are strongly expressed at the plasma membrane in confluent iBREC, but are located in the cytoplasm in non-confluent cells. In the presence of 50 ng/ml VEGF(165), occludin was found in the cytoplasm after 1 to 2 days, whereas claudin-5 was not and ZO-1 was only weakly influenced. However, after addition of 33 microg/ml pegaptanib for 24 h to VEGF(165)-treated iBREC, all tight junction proteins tested were again strongly expressed in the plasma membrane. These results confirm an important role of tight junction proteins in the mechanisms of action of VEGF and pegaptanib on endothelial cells.

Tight junction composition is altered in the epithelium of polycystic kidneys

Kidney cysts in autosomal dominant polycystic kidney disease (ADPKD) undergo progressive enlargement together with luminal fluid secretion. This involves active, uphill transcellular Cl(-) transport which drives passive Na(+) and water secretion. Implicit in this mechanism is the assumption that the paracellular permeability of the cyst epithelium to Cl(-) must be very low. Claudins are tight junction (TJ) transmembrane proteins that determine the ion selectivity of paracellular barriers. The aim of this study was to determine the expression and localization of claudins within renal cysts in a mouse hypomorphic model of ADPKD and in human patients. We found that the majority of cysts were of collecting duct origin. Claudins normally expressed in collecting duct (3, 4, 7, 8, and 10) were found in small cysts. However, only claudin-7 persisted at substantive levels in the dedifferentiated epithelium of large, presumably late-stage cysts, where it was localized both at the TJ and basolaterally. The constitutively expressed TJ proteins, ZO-1 and occludin, were also abundantly expressed and correctly localized, suggesting that the basic infrastructure of the TJ is preserved. A previous study suggested that claudin-7 may function as a paracellular Cl(-) barrier. We postulate that the role of claudin-7 in ADPKD is to seal the paracellular route in Cl(-)-secreting cyst epithelium, preventing backleak of Cl(-), and that it thereby plays a permissive role in fluid secretion and cyst growth.

VEGF-induced effects on proliferation, migration and tight junctions are restored by ranibizumab (Lucentis) in microvascular retinal endothelial cells

Background:Because vascular endothelial growth factor (VEGF) signalling is deregulated in diabetic retinopathy, the potential therapeutic effects of VEGF inhibitors such as the human VEGF-specific antibody ranibizumab are currently being tested....