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PubMed2003

Glucocorticoids control beta-catenin protein expression and localization through distinct pathways that can be uncoupled by disruption of signaling events required for tight junction formation in rat mammary epithelial tumor cells.

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates the remodeling of tight junctions and adherens junctions before formation of highly sealed tight junctions. In this study, the expression and localization of key components of the apical junction were examined as potential targets of glucocorticoid signaling. Western blot and RT-PCR demonstrated that dexamethasone up-regulated beta-catenin protein and transcript expression and nearly ablated beta-catenin phosphorylation under conditions that led to a significant increase in monolayer transepithelial resistance. Indirect immunofluorescence revealed that dexamethasone treatment also caused beta-catenin to localize predominantly at the cell membrane rather than the nucleus. The glucocorticoid regulation of beta-catenin expression and localization was not a consequence of dexamethasone inhibition of cell growth, because both responses were unaltered in the presence of hydroxyurea. The steroid induction of beta-catenin expression and localization can be uncoupled by altering the function of signaling pathways needed for tight junction formation. Expression of dominant-negative RasN17 abolished dexamethasone up-regulation of beta-catenin protein expression without affecting its localization at the membrane. In contrast, exogenous treatment or constitutive production of TGFalpha abolished the dexamethasone-induced alteration of beta-catenin localization without affecting the dexamethasone stimulation of beta-catenin expression. Taken together, our results demonstrate that glucocorticoids control beta-catenin at two distinct levels of cellular regulation that differ in their cell signaling requirements for the glucocorticoid regulation of mammary epithelial junctional dynamics.

Animals↗
PubMed2002

The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line(1).

Chitosan is a polycationic compound widely employed as dietary supplement and also present in pharmaceutical preparations. Although it has been approved for human consumption, its possible side effects have not been widely investigated and the available data in the literature are still controversial. Several polycationic substances have been shown to affect tight junction permeability in epithelial cell models in vitro. In this study we have compared the effects of chitosan and other polycations (polyethylenimine, poly-L-lysines of different molecular weights) on the integrity of tight junctions and of the actin cytoskeleton in the human intestinal Caco-2 cell line. We have measured trans-epithelial electrical resistance and paracellular passage of the extracellular marker inulin, and we have localized F-actin and tight junctional proteins (ZO1 and occludin) in cell monolayers treated with various concentrations of each polycation. Fluorescent poly-L-lysines were also employed to determine their association with the cell monolayer. Our results indicate that all polycations investigated are able to induce a reversible increase in tight junction permeability. This effect is concentration and energy dependent, affected by the extracellular concentration of divalent cations (calcium, magnesium and manganese) and it is associated with morphological changes in the F-actin cytoskeleton, as well as in the localization of tight junctional proteins. Chitosan, in particular, was the only cationic polymer that displayed an irreversible effect on tight junctions at the highest concentration tested (0.01%). These results indicate that oral ingestion of chitosan may have more widespread health effects by altering intestinal barrier function, thus allowing the entrance into the circulation of potentially toxic and/or allergenic substances.

Journal Article↗
PubMed1982

Cytochalasin B affects the gap and tight junctions of mouse hepatocytes in vivo.

This study investigates the effect of cytochalasin B at a dosage of 0.2 mg per mouse per day for a period of 7 days in an in vivo experiment on mouse liver. Using thin-sectioning and freeze-fracture technique both quantitative and qualitative analysis was made of membranes and cell contacts (gap and tight junctions). Significant alterations of both membranes and junctions were observed. The intercellular space showed vacuolar dilatation in some cases and there were vacuoles observed within the cytoplasm. The microvillar bile canaliculi were dilated. However, no colloidal tracer was observed within the lumen following lanthanum perfusion. With the aid of the freeze-fracture method it was possible to demonstrate that the strands of the tight junctions were highly disorganized. In some cases reduction and in other cases proliferation of tight junctions was observed. Large, proliferative plaques of tight junctions were found both in contact with the tight junctions of the bile canaliculus and ending freely on the plasmalemma. The gap junctions appeared enlarged as well. Their average size increased from 0.42 micron 2 to 0.90 micron 2 (p less than 0.005). The enlargement was also accompanied by an increase in the proportion of the plasma membrane occupied by the junctions: 3.42% in control animals, 10.25% in the livers of mice treated with cytochalasin B. Frequently evaginated and internalized gap junctions were seen in the experimental group. In view of the fact that cytochalasin B, in addition to other effects, also has an effect on the microfilament system of the cell, it may be supposed that microfilaments play a role in maintenance of the orderly structure or in the formation of tight and gap junctions. This remains hypothetical, however, and additional studies are necessary in order to conclusively clarify this issue.

Animals↗
PubMed1994

Membrane dynamics during migration of placental cells through trophectodermal tight junctions in sheep and goats.

Binucleate cells in ruminant trophectodermal epithelium are unique in that they form part of the tight junction as they migrate across it, maintaining the ionic barrier seal to the internal milieu of the fetus. Such participation imposes considerable constraints on the cell migration because membrane cannot flow through a tight junction. We report quantitative ultrastructural immunocytochemical evidence for vesicle membrane insertion into the binucleate cell plasmalemma which allows the cells to form a pseudopodium past the tight junction. This pseudopodium increases continuously in area by vesicle insertion and develops a close apposition to the plasmalemma of the fetomaternal syncytium which constitutes the fetomaternal boundary in the placenta of the sheep and goat. Eventually the apposed membranes of the binucleate cell pseudopodium and the syncytium fuse by vesiculation and the cytoplasm and nuclei of the binucleate cell merge into the fetomaternal syncytium. The binucleate cell plasmalemma remaining on the trophectodermal side of the tight junction is blebbed off into, and phagocytosed by, the uninucleate trophectodermal cells between which the binucleate cell passed. This process permits the delivery of the binucleate cell granules to the maternal side of the placenta but none of the fetal molecules expressed on the plasma membrane of the binucleate cells are exposed to potential maternal immunological rejection.

Animals↗
PubMed1997

Occludin as a possible determinant of tight junction permeability in endothelial cells.

Endothelial cells provide a crucial interface between blood and tissue environments. Free diffusion of substances across endothelia is prevented by the endothelial tight junction, the permeability of which varies enormously depending on tissue. Endothelial cells of the blood-brain barrier possess tight junctions of severely limited permeability, whereas those of non-neural tissue are considerably leakier, but the molecular basis for this difference is not clear. Occludin is a major transmembrane protein localizing at the tight junction. In this study, we show, by immunocytochemistry, that occludin is present at high levels and is distributed continuously at cell-cell contacts in brain endothelial cells. In contrast, endothelial cells of non-neural tissue have a much lower expression of occludin, which is distributed in a discontinuous fashion at cell-cell contacts. The apparent differences in occludin expression levels were directly confirmed by immunoblotting. The differences in occludin protein were reflected at the message level, suggesting transcriptional regulation of expression. We also show that occludin expression is developmentally regulated, being low in rat brain endothelial cells at postnatal day 8 but clearly detectable at post-natal day 70. Our data indicate that regulation of occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues.

Amino Acid Sequence↗
PubMed2005

Na+/K+ -ATPase regulates tight junction formation and function during mouse preimplantation development.

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment on the distribution of ZO-1 and occludin (tight junction associated proteins). Ouabain treatment (up to 6 h) or culture in K(+)-free medium (up to 6 h) resulted in the appearance of a discontinuous ZO-1 protein distribution and a loss of occludin immunofluorescence. The third set of experiments examined the influence of ouabain treatment on tight junction function. Ouabain treatment or culture in K(+)-free medium affected tight junction permeability as indicated by an increase in the proportion of treated embryos accumulating both 4 kDa and 40 kDa fluorescein isothiocyanate (FITC)-dextran into their blastocyst cavities. The results indicate that the Na(+)/K(+)-ATPase is a potent regulator of tight junction formation and function during mouse preimplantation development.

Animals↗
PubMed2002

Absence of tight junction formation in an allogeneic graft cell line used for developing an engineered artificial salivary gland.

An essential structural feature of fluid-secreting epithelial tissues is the presence of tight junctions. To develop a tissue-engineered organ capable of fluid secretion, the cellular component must establish these structures. As part of efforts to create an engineered artificial salivary gland, we have examined the ability of a candidate allogeneic graft cell line, HSG, to produce several key tight junction proteins, as well as to exhibit functional activities consistent with effective tight junction strand formation. In contrast to results obtained with a control kidney cell line, MDCK-II, HSG cells were unable to synthesize four important tight junction-associated proteins: ZO-1, occludin, claudin-1, and claudin-2. In addition, unlike MDCK-II cells, HSG cell monolayers could not restrict paracellular permeability. HSG cells were, thus, unable to generate significant transepithelial electrical resistance or serve as an effective barrier to osmotically imposed fluid movement. Furthermore, these two functional activities could not be reconstituted via the stable transfection of HSG cells with cDNAs encoding either claudin-1 or claudin-2. We conclude that because of their inability to form tight junctions, HSG cells are unsuitable for use as an allogeneic graft cell in an artificial salivary fluid secretory device. These studies also emphasize the importance of graft cell selection in artificial organ development, as certain required characteristics may be difficult to reengineer.

Cell Line↗
PubMed1996

Loss of occludin and functional tight junctions, but not ZO-1, during neural tube closure--remodeling of the neuroepithelium prior to neurogenesis.

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.

Amnion↗
PubMed1999

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

Amino Acid Sequence↗
PubMed1995

Tight junctions and the molecular basis for regulation of paracellular permeability.

Tight junctions create a regulated paracellular barrier to the movement of water, solutes, and immune cells between both epithelial and endothelial cells. Recent progress has been made in identifying the proteins that create this barrier. The transmembrane protein occludin is an excellent candidate for the sealing protein and is bound on the cytoplasmic membrane surface to the proteins ZO-1 and ZO-2. Functions for ZO-1 and ZO-2 are suggested by their invertebrate homologues, one of which is a tumor suppressor and another is required in epidermal growth factor receptor signaling. Multiple cellular signaling pathways affect assembly and sealing of junctions. Dynamic regulation of perijunctional actin has emerged as a unifying hypothesis for controlling paracellular permeability. Understanding and manipulating permeability will require a more detailed molecular characterization of tight junction proteins and in particular a characterization of how cell signaling regulates their attachment to the perijunctional cytoskeleton.

Actins↗
PubMed1975

Changes in tight junctions of thyroid epithelium with changes in thyroid activity.

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated. In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects. Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions. Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.

Animals↗
PubMed1987

Tight junctions of brain endothelium in vitro are enhanced by astroglia.

The belts of endothelial tight junctions, which impede diffusion between blood and brain, were reduced to fragmentary, small junctions in subcultured brain endothelium. When cocultured with the capillaries' nearest neighbor, the astrocytes, these endothelial tight junctions were enhanced in length, width, and complexity, as seen by en face views of the cell membranes with freeze-fracture electron microscopy. Gap junctions, common in brain endothelium in vitro but absent in mature brain capillaries in vivo, were markedly diminished in area from among the enhanced tight junctions of the cocultures. Thus, astrocytes in vitro play a role in the formation, extent, and configuration of the junctional complexes in brain endothelium, whose diffusion barrier may likewise be influenced by astrocytes in vivo.

Animals↗
PubMed2002

Calcium modulation of adherens and tight junction function: a potential mechanism for blood-brain barrier disruption after stroke.

BACKGROUND: This review deals with the role of calcium in endothelial cell junctions of the blood-brain barrier (BBB). Calcium is critical for adherens junction function, but it appears that calcium is also important in regulating tight junction function necessary for the barrier characteristics of cerebral microvessels. SUMMARY OF REVIEW: The BBB is critical for brain homeostasis and is located at the cerebral microvessel endothelial cells. These endothelial cells maintain their barrier characteristics via cell-cell contacts made up of adherens and tight junctions. Adherens junctions are calcium dependent; recent evidence suggests that calcium also affects tight junctions. After stroke, there is a disruption of the BBB. Interfering with calcium flux under hypoxic conditions can prevent BBB breakdown. Calcium may alter BBB junction integrity by a number of different signal transduction cascades, as well as via direct interaction of calcium ions with junction proteins. It remains to be determined whether clinical use of calcium channel antagonists is a viable means to reduce BBB disruption after stroke. CONCLUSIONS: With the widespread use of calcium channel blockers as clinical treatments for hypertension, which is a risk factor for stroke, the exact role of calcium in modulating BBB integrity needs to be elucidated.

Adherens Junctions↗
PubMed1998

Effect of cytokines and nitric oxide on tight junctions in cultured rat retinal pigment epithelium.

PURPOSE: These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro. METHODS: Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin. RESULTS: Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining. CONCLUSIONS: Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.

Actins↗
PubMed2005

Modification of epithelial tight junction integrity to enhance transmucosal absorption.

Tight junctions (TJs) located between adjacent epithelial cells restrict the movement of solutes, ions, and even water. Restriction of macromolecules by TJ structures impedes one potential route of absorption for biopharmaceutical drugs, a route known as the paracellular pathway. It is anticipated that an improved understanding of TJ structure and cellular mechanisms that regulate its function will lead to successful strategies for improved transmucosal drug absorption through the paracellular pathway. Based upon clinical correlations between TJ dysfunction and several disease states, any approach to modify TJ integrity to enhance drug absorption in this way must be tempered by an appreciation of potential safety concerns that might develop through sustained or repeated TJ alterations. This review describes a current understanding of epithelial TJ organization and examines potential methods and implications for regulated modification of TJ function for the enhanced uptake of drugs without detrimental clinical outcomes.

Amino Acid Sequence↗
PubMed1986

Effects of aspirin on tight junction structure of the canine gastric mucosa.

The effects of aspirin on the canine gastric mucosal barrier were examined using the freeze-fracture and extracellular tracer techniques. Aspirin treatment (3, 20, or 40 min) resulted in alterations in tight junction complex morphology and permeability. Discontinuities in the apical occluding complex, hyperplastic tight junctions (extensions of the apical tight junction strands radiating over the lateral plasma membrane), and a variability in the number of strands (1-20) comprising the complex were observed. A concurrent increase in lanthanum permeability between nonnecrotic surface mucous epithelial cells was also demonstrated. The results of these experiments may suggest that aspirin-induced impairment of the tight junction complexes between viable gastric mucosal epithelial cells may be a major contributing factor in the etiology of stomach disorders.

Animals↗
PubMed2005

Changes in tight junctional resistance of the cervical epithelium are associated with modulation of content and phosphorylation of occludin 65-kilodalton and 50-kilodalton forms.

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (R(TJ)). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in R(TJ) induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of R(TJ) correlated with the 65-kDa form, whereas low levels of R(TJ) correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the R(TJ), 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

Cell Line, Tumor↗
PubMed2005

Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption.

Chitosan has been successfully used as an excipient for trans-epithelial drug delivery systems. It is known to transiently open intercellular tight junctions thus increasing the permeability of an epithelium. In order to investigate the possible role of protein kinases in trans-epithelial delivery, changes in trans-epithelial electrical resistance ('TEER') of epithelial (Caco-2) cell monolayers were assessed in response to chitosan glutamate treatment, in the presence and absence of specific protein kinase inhibitors. Changes in subcellular localisation of the tight junction protein ZO-1 observed by immunofluorescence and western blotting of cellular fractions were also assessed. Inhibition of protein kinase C (PKC), but not mitogen activated protein kinase (MAPK) was found to prevent the chitosan-mediated decrease in TEER, and changes in localisation of ZO-1. In order to determine which PKC isozymes were responsible for the chitosan-mediated tight junction disruption, the activation of the PKC isozymes alpha, beta and delta was investigated. A chitosan-mediated translocation of PKC alpha but not PKC beta or delta from the cytosol to the membrane fraction, indicative of PKC alpha activation was observed. Thus, treatment of Caco-2 cells with chitosan may result in the activation of PKC-dependent signal transduction pathways which affect tight junction integrity.

Animals↗