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PubMed1998

ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin.

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19-amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

Amino Acid Sequence↗
PubMed2003

Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions.

A recent study (Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C., and Mrsny, R. J. (2000) J. Biol. Chem. 275, 29816-29822) suggested that phosphatidylinositol 3-kinase (PI 3-kinase) may interact with occludin; however, there exists no evidence of direct interaction of PI 3-kinase with the tight junctions. Activation of PI 3-kinase by oxidative stress and its role in disruption of tight junctions was examined in Caco-2 cell monolayer. The oxidative stress-induced decrease in electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 were reduced by a PI 3-kinase inhibitor, LY294002. Oxidative stress-induced tyrosine phosphorylation and dissociation from the actin cytoskeleton of occludin and ZO-1 were reduced by LY294002. The regulatory subunit of PI 3-kinase, p85, and the PI 3-kinase activity were co-immunoprecipitated with occludin, which were rapidly increased by oxidative stress. Oxidative stress resulted in increased translocation of p85 from the intracellular compartment into the intercellular junctions. Pair-wise glutathione S-transferase pull-down assay showed that glutathione S-transferase-occludin (C-terminal tail) binds to recombinant p85. This study shows that oxidative stress increases the association of PI 3-kinase with the occludin, and that PI 3-kinase activity is involved in oxidative stress-induced disruption of tight junction.

Chromones↗
PubMed1985

Functional analysis of tight junction organization.

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.

Animals↗
PubMed1979

Gap and tight junctions in tunicates. Study in conventional and freeze-fracture techniques.

Intercellular junctions have been investigated in epidermis and pharyngeal epithelium of larvae and adults of various species of tunicates with conventional and freeze-fracture techniques. Gap and tight junctions were found, similar to those observed in vertebrate tissues. Gap junctions were frequent in glandular epithelia and in larval tissues. They were interpreted as ways of intercellular communication in these developing tissues. They were also particularly numerous in Phallusia pharyngeal cells. Tight junctions were found preferentially in adult pharyngeal and epidermal epithelia, where they were arranged in strands of distinct particles forming a belt-like network at the apical part of cells. These junctions were interpreted as providing a tight barrier between the internal medium and the external environment. In larvae, tight junctions were found only between epidermal cells of the tail. These junctions thus characterized completely differentiated tissues, where they might play, in tunicates and in vertebrates, the same role as septate junctions do in invertebrates.

Animals↗
PubMed1998

Tight junctions and the experimental modifications of lipid content.

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms.

Animals↗
PubMed1999

Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells.

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.

Actins↗
PubMed1993

The making of a tight junction.

MDCK (epithelial cells from the dog kidney) plated at confluence, establish tight junctions in 12-15 hours through a process that requires protein synthesis, formation of a ring of actin filaments in close contact with the lateral membrane of the cells, calmodulin, and a Ca(2+)-dependent exocytic fusion of tight junction (TJ)-associated components. Monolayers incubated in the absence Ca2+ make no TJs. Yet, if Ca2+ is added under these circumstances, TJs are made with a faster kinetics. Ca2+ is needed mainly at a site located on the outer side of the cell membrane, where it activates uvomorulin and triggers the participation of the cellular components mentioned above, via G-proteins associated with phospholipase C and protein kinase C. In principle, the sites of all these molecules and mechanisms involved in junction formation may be where a variety of agents (hormones, drugs, metabolites) act to produce epithelia with a transepithelial electrical resistance (TER) ranging from 10 to 10,000 omega.cm2. This range may be also due to a variety of substances found in serum and in urine, that increase the TER in a reversible and dose-dependent manner.

Actins↗
PubMed1983

Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure.

In some epithelia, mucosal exposure to osmotic loads produces an increase in transepithelial resistance that is presumed to relate to the collapse of the paracellular spaces. Since proximal small intestinal epithelium may transiently encounter osmotic loads during normal digestion, we examined the short-term effect of osmotic loads on resistance and on epithelial structure of mucosal sheets prepared from guinea pig jejunum using Ussing-chamber, thin-section electron-microscopic, and freeze-fracture techniques. After equilibration of mucosal sheets in chambers, mucosal buffer tonicity was increased to 600 mosM with mannitol. This resulted in a 64% increase in resistance within 20 min. Concomitantly, 600 mosM produced a decrease in tight-junction cation selectivity as judged from dilution potentials, collapse of paracellular spaces, decreased cytoplasmic electron density in 10-40% of absorptive cells, and focal absorptive-cell subjunctional lateral-membrane evaginations often associated with microfilament arrays. Freeze-fracture replicas of absorptive-cell tight junctions revealed significant increases in both strand count and depth. Preincubation with 5 micrograms/ml cytochalasin D reduced the 600 mosM resistance increase caused by 600 mosM exposure by 48% but did not prevent the collapse of paracellular spaces. Lowered temperatures that produced morphologic evidence consistent with a gel-phase transition of absorptive-cell lateral membranes prevented both the resistance response and the alterations in tight-junction structure. In conclusion, transient osmotic loads produce an increase in resistance in jejunal epithelium and alter both absorptive-cell tight-junction charge selectivity and structure. These responses, which may have physiologic implications, can be reduced by cytoskeletal inhibitors and ablated by conditions that restrict mobility of absorptive-cell lateral-membrane molecules.

Animals↗
PubMed2004

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

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

Animals↗
PubMed2001

Na,K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells.

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na(+) homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.

Animals↗
PubMed1998

Role of tight junctions in establishing and maintaining cell polarity.

The tight junction (TJ) is not randomly located on the cell membrane, but occupies a precise position at the outermost edge of the intercellular space and, therefore, is itself considered a polarized structure. This article reviews the most common experimental approaches for studying this relationship. We then discuss three main topics. (a) The mechanisms of polarization that operate regardless of the presence of TJs: We explore a variety of polarization mechanisms that operate at stages of the cell cycle in which TJs may be already established. (b) TJs and polarity as partners in highly dynamic processes: Polarity and TJs are steady state situations that may be drastically changed by a variety of signaling events. (c) Polarized distribution of membrane molecules that depend on TJs: This refers to molecules (mainly lipids) whose polarized distribution, although not the direct result of TJs, depends on these structures to maintain such distribution.

Animals↗
PubMed2004

[GERD and tight junction proteins of the esophageal mucosa].

Recent advancement in the research of GERD has revealed that endoscopy negative GERD may not be a milder form of erosive GERD and may have different pathogenesis. We have previously proven that hypersensitivity to the acid of the esophageal mucosa plays an important role in its pathogenesis. Regarding the mechanisms for the esophageal hypersensitivity, we hypothesized that the tight junction proteins of the esophageal mucosa are fully or partially impaired in GERD patients. Accordingly, we immunohistologically studied the expression of various tight junction proteins using the rat reflux esophagitis model. The results demonstrated that the several kinds of tight junction proteins are expressed differently in the various parts of esophagus and their expression altered according to the development of reflux esophagitis.

Animals↗
PubMed2003

Pulses of cell Ca(2+) and the dynamics of tight junction opening and closing.

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca(2+) levels. In this model, TJs were assumed to be specifically controlled by the Ca(2+) concentration levels at the extracellular Ca(2+) binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca(2+) concentration (Ca(2+) (cell)), which might result from changes \ of extracellular Ca(2+) concentration (Ca(2+) (extl)), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca(2+)-switch experiments were performed in which changes of Ca(2+) (cell) were induced using the Ca(2+) ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca(2+)-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance ( G), showing that the sealing of TJs is not affected by a rise in Ca(2+) (cell). When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca(2+) (cell) never led to a decline of G, indicating that a rise of Ca(2+) (cell) does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca(2+) (cell) is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca(2+) (cell) is without effect on the TJs, since an increase of Ca(2+) (cell) not only causes a transient increase of G but, in addition, during oscillations a rise of Ca(2+) (cell) induced by the Ca(2+) ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca(2+) concentration (Ca(2+) (bl)) is increased after TJs were opened by Ca(2+) (bl) removal cannot be ascribed to a rise of Ca(2+) (cell) and might be a consequence of Ca(2+) binding to extracellular Ca(2+) sites.

Animals↗
PubMed2004

Developmental changes in proximal tubule tight junction proteins.

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

Age Factors↗
PubMed2001

Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions.

Acetaldehyde-induced cytotoxicity is an important factor in pathogenesis of alcohol-related diseases; however, the mechanism of this toxicity is unknown. We recently showed that acetaldehyde increases epithelial paracellular permeability. We asked whether protein tyrosine phosphorylation via modulation of tyrosine kinases and/or PTPases is a mechanism involved in acetaldehyde-induced disruption of the tight junctions in the Caco-2 cell monolayer. Immunofluorescence localization of occludin and ZO-1 showed disruption of the tight junctions in acetaldehyde-treated cell monolayer. Administration of genistein prevented acetaldehyde-induced permeability. Acetaldehyde increased tyrosine phosphorylation of three clusters of proteins with molecular masses of 30-50, 60-90, and 110-150 kDa; three of these proteins were ZO-1, E-cadherin, and beta-catenin. Acetaldehyde reduced PTPase activity in plasma membrane and soluble fractions, whereas tyrosine kinase activity remained unaffected. Treatment with acetaldehyde resulted in a 97% loss of protein tyrosine phosphatase (PTP)1B activity and a partial reduction of PTP1C and PTP1D activities. These results strongly suggest that acetaldehyde inhibits PTPases to increase protein tyrosine phosphorylation, which may result in disruption of the tight junctions.

Acetaldehyde↗
PubMed2002

Tight junction proteins: a novel class of integral membrane proteins. Expression in human epidermis and in HaCaT keratinocytes.

Tight junction proteins comprise a novel group of integral membrane proteins necessary for cell-to-cell contacts and responsible for the barrier function in epithelial and endothelial cells in various tissues. The tight junction membrane domain contains at least three distinct proteins, named occludin, claudin and junctional adhesion molecule. Claudins are products of a gene family consisting of more than 20 members. We investigated mRNA expression of occludin and 13 different claudins in neonatal foreskin, adult skin and cultivated HaCaT keratinocytes by the Northern blot technique, and performed immunohistochemical staining of adult skin for occludin, claudin 1 and claudin 2. Occludin, claudin 1 and claudin 3 mRNAs were expressed in human neonatal and adult keratinocytes as well as in HaCaT keratinocytes. All other tested claudins were negative. Immunohistochemical staining of adult skin was positive for occludin in the intercellular space of the granular layer, and for claudin 1 in the inter-cellular space of the spinosum layer and basal layer, but negative for claudin 2 in all skin layers. Claudin 1 was also positive in the outer root sheath of hair follicles. Our results indicate that occludin, claudin 1 and claudin 3 are involved in cell-to-cell contacts between keratinocytes in human epidermis, although their functional importance remains unknown.

Adult↗
PubMed2002

Polarized distribution of carcinoembryonic antigen is associated with a tight junction molecule in human colorectal adenocarcinoma.

This study we presents a novel anti-occludin monoclonal antibody that can be used for formalin-fixed, paraffin-embedded tissue sections. The relationships between aberrant localization of carcinoembryonic antigen (CEA) and abnormalities of tight junctions were studied in human colorectal cancers by this antibody. Abnormalities in the cell surface expression of CEA have been shown to be characteristic of human colorectal cancer cells. Cancer cells that participated in the formation of glandular structures expressed occludin at the apical cell border and CEA was expressed more apically than occludin. Where cancer cells showed solid nests without glandular structures, occludin was completely lost and CEA was demonstrated in a diffuse pattern throughout the cells. These findings suggest that the polarized apical expression of CEA in neoplastic glandular structures depends on the expression of occludin and the fence function of tight junctions. During tumour progression, loss of occludin may lead to the loss of membrane polarity and the non-polarized expression of CEA. The antibody described provides a powerful tool for the study of tight junctions in surgically resected human tissue.

Adenocarcinoma↗
PubMed1980

Formation of gap and tight junctions between reaggregated blastomeres of the killifish, Fundulus.

Blastomeres from eggs of the killifish, Fundulus, were mechanically dissociated and reaggregated by pelleting in a simple saline solution. Formation of gap and tight junctions was followed by electron microscopy of freeze-fracture replicas. Five to eight min after pelleting, neither new nor old junctions were observed. After 10-15 min, small gap junctions were found, but these were not associated with distinct formation plaques. Larger gap junctions were observed after 45 min, and the images were consistent with growth by accretion of intramembrane particles. In aggregates, after 20 min or more, tight junctions were much more commonly found than in intact blastulae, and it seemed likely that they were being formed by cells that were not doing so in the intact embryo. Initial stages consisted of short strands that appeared to grow in length. Also, more elaborate junctions were seen than occur in situ. Particle-free membrane often occurred near incomplete junctions, and large junctions like those in situ separated particle-rich from particle-free membrane. In this system, the formation of both gap and tight junctions occurs with shorter latency, and is more precisely timed, than heretofore described.

Animals↗