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PubMed1999

Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin.

The effects of interferon-gamma (IFN-gamma) on tight junctions in T84 human intestinal epithelial cells were investigated. Treatment of T84 cells with IFN-gamma caused a dose- and time-dependent increase in monolayer permeability as assessed by transepithelial electrical resistance measurements. Examination of specific proteins associated with tight junctions by immunoblotting and confocal microscopy revealed changes in the expression levels and localization of some of these proteins after exposure of the cells to IFN-gamma. Specifically, IFN-gamma treatment resulted in an almost total loss of zonula occludens (ZO)-1, whereas the levels of ZO-2 and occludin showed relatively modest decreases compared with untreated cells. Loss of ZO-1 was associated with the altered localization of ZO-2 and occludin. In IFN-gamma-treated cells, ZO-2 and occludin were diffusely distributed, whereas, in control cells, they, along with ZO-1, were predominantly localized to the tight junctions. Analysis of ZO-1 protein and RNA by pulse chase and RT-PCR, respectively, showed an increase in protein turnover, a decrease in protein synthesis, and a reduction in RNA levels following IFN-gamma treatment. In contrast to ZO-1, ZO-2 and occludin did not show any major changes in these parameters. In addition, the organization of actin in the apical and tight junction regions, but not in the mid- or basal regions, of the cells was also perturbed by IFN-gamma treatment of cells. Time-course analysis of IFN-gamma-induced alterations in ZO-1 expression and apical actin perturbation indicated that these two effects were intimately linked and could not be dissociated. These results suggest that IFN-gamma affects barrier function in T84 cells by decreasing the levels of ZO-1 and perturbing apical actin organization, which leads to a disorganization of the tight junction and an increase in paracellular permeability.

Actins↗
PubMed1984

A study of microvasculature in normal and inflammatory synovial membranes in the rabbit using light and electron microscopy and freeze fracture.

The Authors made a study of microvasculature in normal and inflammatory synovial membranes in the rabbit, by light and electron microscopy and with the use of freeze fracture. The synovial membrane of the knee was studied in 15 normal rabbits and in 22 rabbits in whom immunization with mycobacterium tuberculosis crude cytoplasmic water soluble extract had provoked inflammatory synovitis. A comparison between normal and inflammatory synovia by light microscopy showed, in inflammatory synovitis, a significant increase in the number of congested and obliterated microvessels (p less than 0.001) and in the number of endothelial nuclei per microvessel (p less than 0.0001). By electron microscopy no specific alteration of synovial microvessels was observed. A significant increase was found in the number of venules in inflammatory synovitis (p less than 0.01). The number of simple interendothelial tight junctions, defined by 1 or 2 interendothelial contacts, and of complex interendothelial tight junctions, defined by 3 or more interendothelial contacts, was similar in venules and in capillaries of inflammatory synovitis and of normal controls. The mean number and mean spacing of junctional strands in interendothelial tight junctions was similar in the microvessels of inflammatory synovitis and of normal controls. The Authors conclude that inflammation does not provoke specific alterations in synovial microvasculature but could induce an adaptative state resulting in increased synovial permeability.

Animals↗
PubMedDate not supplied

Freeze fracturing of the human stria vascularis.

The stria vascularis is an important functional element in the mammalian cochlea. This special tissue is considered to be the source of the endocochlear potential and thus the driving force for the production of a receptor response to the auditory stimulus. In order to maintain its function, the stria vascularis needs to be separated from the endolymphatic space by a tight seal. This seal is comprised of tight junctions in the marginal cell layer. The junctional arrangement in the stria vascularis is described, utilizing the freeze-fracturing technique which allows the visualization of large expansions of plasma membrane. The marginal cells are generally separated by tight junctions of the moderately tight to tight type. In places, however, even so-called leaky junctions with only a few sealing strands are present. Whereas the intermediate cell layer seems to lack tight junctions, the basal cells are connected by extensive tight junctions more or less covering the entire cell. These junctions seem to form an extremely tight barrier against the spiral ligament. Gap junctions are also present in the tissue. Intermediate cells as well as the basal cells are coupled by gap junctions. In the basal cell layer, gap junctional elements may also be found inside the large tight junctions comprising so-called mixed junctions.

Adult↗
PubMed2003

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice.

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.

Actins↗
PubMed1977

Cohesion of horny cells during comedo formation. An electron microscope study.

Cohesion between horny cells was studied in experimentally induced comedones, from the rabbit's external ear canal. It occurred in two different ways: initially, by the persistence of desmosones and desmonosmal bodies (fusiform electron dense bodies), which loosely held the horny cells together; later, and to a lesser extent, by tight junctions, which tightly bound the horny cells together. A gradual decrease in the numbers of membrane coating granules was observed.

Acne Vulgaris↗
PubMed1998

Clinical models of intestinal adaptation.

Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na-glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self-filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na-glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor-alpha can specifically downregulate the tight junction, as indicated in the intestinal HT-29/B6 cell model.

Animals↗
PubMed2004

IL-1beta regulates expression of Cx32, occludin, and claudin-2 of rat hepatocytes via distinct signal transduction pathways.

The functions of gap and tight junctions are perturbed during the acute-phase response to liver injury. To elucidate the mechanism of pro-inflammatory cytokine IL-1beta responsible for regulation of hepatic gap and tight junctions, we analyzed expression and function of gap and tight junctions using a rat liver injury model and primary cultures of rat hepatocyte. In rat liver lobules at 24 h after thioacetamide (TAA) treatment, where some IL-1beta-positive non-parenchymal cells existed, disappearance of connexin32-positive spots at cell borders of the hepatocytes and increases of claudin-2 and occludin immunoreactivities in bile canalicular regions were observed. In primary cultures of rat hepatocytes, IL-1beta caused the disappearance of connexin32, which was reciprocal to the induction and localization of claudin-2 to cell membranes. The downregulated connexin32 expression was inhibited by treatment with a MAP-kinase inhibitor (PD98059), whereas the upregulated claudin-2 expression was blocked by p38 MAP and PI3-kinase inhibitors (SB203580 and LY294002). The changes of connexin32 and claudin-2 may be controlled at the transcriptional level via NF-kappaB, HNF-1alpha, and CDX2. Occludin was hyperphosphorylated by IL-1beta treatment and was inhibited by treatment with a PI3-kinase inhibitor. These results demonstrate that MAP-kinase, p38 MAP-kinase, and PI3-kinase are distinctly involved in the regulation of hepatic gap and tight junctions during the acute-phase response to IL-1beta.

Animals↗
PubMed1991

Transport of HRP through Reissner's membrane in experimental endolymphatic hydrops.

Unilateral endolymphatic hydrops was produced in guinea pigs by cauterization of the endolymphatic sac. Measurements of compound action potential (CAP), cochlear microphonics (CM) and negative summating potential (-SP) confirmed endolymphatic hydrops three months after surgery. In both control and hydropic ears, reaction product of HRP was observed only on the perilymphatic surface of the epithelial cells of Reissner's membrane after 10 min perfusion, while it was observed on both the endolymphatic and perilymphatic surfaces after 30 min perfusion. Epithelial tight junctions were not stained and labelled pinocytotic vesicles were observed in the epithelial cells. These findings suggest that the transport of HRP through Reissner's membrane is unchanged in endolymphatic hydrops and that the epithelial junctions are tight regardless of the distension of Reissner's membrane.

Animals↗
PubMed1984

Capillary junctions of the rat are not affected by osmotic opening of the blood-brain barrier.

Osmotic opening of the blood-brain barrier had no effect on the structure of the interendothelial tight junctions located within approximately 9 micron 2 of brain capillary endothelial plasma membrane (junction-containing) examined in this study. These tight junctions restrict the passive diffusion between the blood and the brain and constitute the anatomic basis of the blood-brain barrier. Increased permeability of the blood-brain barrier in the cerebral cortex of the right hemisphere of rats, induced by an infusion of a hypertonic solution of arabinose and monitored with the protein tracer horseradish peroxidase (HRP), was evidenced by the extravasation of the tracer into the extracellular compartment of the brain. Freeze-fracture analysis of the capillaries from the same tissue revealed no alterations in the intramembrane components of the endothelial tight junctions. The junctions, which consist of 8-12 highly anastomosed parallel ridges situated on the PF fracture face of the endothelial plasmalemma, showed no loss of ridge continuity or intra-ridge connections, and were identical to zonulae occludentes from control capillaries. Consistent labeling of numerous vesiculo-tubular elements by HRP in the endothelia of experimental tissue and the three-dimensional nature of these elements observed in platinum replicas support the interpretation that these structures represent transendothelial conduits which are continuous with the luminal and abluminal surfaces of the endothelial cells. Absence of similar structures in control endothelia is taken as evidence that their presence in experimental tissues is a direct response to the osmotic insult. It was concluded, therefore, that during osmotic opening of the blood-brain barrier passage of HRP across the endothelium of brain capillaries is not by an inter-endothelial route due to disruption of tight junctions but rather by a transendothelial route due to amplified vesicular activity.

Animals↗
PubMed1997

Mammalian occludin in epithelial cells: its expression and subcellular distribution.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions, and the cDNA encoding its mammalian homologue was identified very recently (Ando-Akatsuka, Y., M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, Sh. Tsukita, J. Cell Biol. 133, 43-47 (1996)). Here we describe the basic properties of mammalian occludin in epithelial cells at the DNA, RNA, and protein levels. The human occludin gene was mapped to chromosome band 5q13.1 by fluorescent in situ hybridization. Northern blotting identified several occludin mRNA bands, suggesting the possible expression of several alternatively spliced products. Occludin mRNA was detected in cultured epithelial cells, but not in cultured fibroblasts. The mRNA level was high in the testis, kidney, liver, lung, and brain, which reportedly bear well-developed tight junctions. We then produced monoclonal and polyclonal antibodies using recombinant mouse occludin as the antigen, which reacted not only with mouse, but also human, dog and pig occludin. These antibodies recognized several bands around 60 kDa in epithelial cells but not in fibroblasts. Immunofluorescence microscopy of various tissues revealed that the staining intensity of occludin correlated well with the number of tight junction strands in epithelial cells. By contrast, the staining of ZO-1, a well-characterized tight junction-associated protein, was not specific for tight junctions. Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy.

Animals↗
PubMed1998

Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation.

We have examined freeze-fracture replicas of maxillary first molar tooth germs of newborn rats at early stages of dentinogenesis to study the development of tight junctions in the distal plasma membrane of differentiating odontoblasts. In addition, freeze-fracture was combined with filipin to observe the distribution of cholesterol on the distal plasma membrane of odontoblasts during differentiation. Only gap junctions were present in early differentiating odontoblasts. The distal plasma membrane exhibited low cholesterol content, which might indicate high fluidity. With the beginning of mineral deposition in matrix-vesicles, the first signs of tight junction formation were observed. Further development revealed increasingly complex focal tight junctions. In later stages, when mineralisation is observed progressing to the fibrillar and non-fibrillar constituents of the matrix, well developed focal tight junctions were detected. Concomitantly, cholesterol in distal portions of the odontoblast plasma membrane increased, indicating, probably, a higher rigidity. Thus, a distal plasma membrane domain is established, odontoblasts become fully differentiated, and partial compartmentalisation of matrix occurs. At this stage, odontoblasts may be able to secrete specific matrix molecules to ensure the progression of mineralisation.

Animals↗
PubMed2005

Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells.

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 A in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.

Animals↗
PubMed2003

Age-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1).

The endothelial or epithelial tight junctions create a rate-limiting barrier to diffusion of solutes. A major determinant of the barrier function is the density of tight junction proteins. Since aging is associated with significant alterations in the blood-brain barrier (BBB) it is possible that specific tight junction proteins may be altered in the cerebrum of aging rats. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and their mRNA in cerebral tissue of 3-, 12- and 24-month-old rats. The cerebral occludin content in 24-month-old rats (732.5+/-99.9 arbitrary units) was significantly reduced compared to 12-month-old rats (1043.4+/-131.8) or 3-month-old rats (1021.4+/-62.8), P<0.01. The cerebral ZO-1 protein content in 24-month-old rats (161.7+/-8.1 arbitrary units) and 12-month-old rats (144.3+/-35.9) were not significantly reduced compared to 4-month-old rats (189.0+/-27.2). The occludin mRNA content relative to G3PDH mRNA was 1.11+/-0.05, 1.11+/-0.07 and 1.00+/-0.05 in 3-, 12- and 24-month-old rats, respectively. The differences did not achieve statistical significance. The ZO-1 mRNA content of cerebral tissue relative to G3PDH mRNA was significantly increased in 24-month-old rats compared to 3-month-old rats (1.280+/-0.030 vs. 0.956+/-0.038), P<0.001. It is concluded that aging in rats may alter the molecular anatomy of the BBB by altering the content of select structural proteins of tight junctions.

Aging↗
PubMed1978

Morphologic changes of the junctional complex of the hepatocytes in rat liver after bile duct ligation.

Using freeze-fracture techniques, we have examined the changes in the morphology of the tight junctional network around the canalicular lumen of the hepatocytes in rat liver after experimental bile-duct ligation. The more or less regular belt of parallel strands formed by the tight junctions around the canalicular lumen of normal hepatocytes is changed after extrahepatic obstruction. A more irregular network is formed withe a reduced number of strands which also extend more in an abluminal direction with formation of irregular loops. Striking changes are seen at the gap junctions: the small patches normally situated within the tight junctional network become less numerous; at some canaliculi they are even absent; also the larger gap junctional areas normally present in deeer abluminal extensions of the lateral cell membrane become hard to find or are even absent. This altered tight junctional pattern suggests an increased permeability so that the pathway of intercellular escape of biliary constituents towards the blood stream in cholestasis becomes as plausible as transhepatocytic regurgitation. The disappearance of the gap junctions would result in a lack of intercellular communication and uncoupling of liver cells, which may lead to a more individual behaviour of adjecent hepatocytes, explaining the heterogeneity in canalicular changes in cholestasis.

Animals↗
PubMed1985

Diffusion barriers in the vaginal epithelium during the estrous cycle in guinea pigs.

In the present study the permeability barriers of the multilayered vaginal epithelium were examined using tracer perfusion techniques, freeze-fracture and thin sectioning. During diestrus and proestrus the upper layers of mucified epithelial cells exhibit tight-junctional belts, which restrict tracer molecules such as lanthanum and horseradish peroxidase. When the highly mucified cells begin to degenerate toward the end of proestrus the underlying epithelium is already keratinized as typical for estrus. The keratinized epithelial cells have a tight-junctional network that joins the basal plasma membranes with the apical membranes of subjacent cells and blocks paracellular diffusion of the tracer molecules. During conversion of the cornified epithelium to a mucified epithelium in metestrus the intercellular space of the epithelium is stained by tracer molecules even though tight-junctional belts can be observed. These results indicate that during cyclic changes of the vaginal epithelium tight junctions can, in general, be considered for the restriction of paracellular diffusion. In metestrus, however, junctions become functionally leaky although they remain morphologically intact. Intercellular lipids, which are normally common in cornified epithelia, are extremely rare and cannot constitute an effective barrier to diffusion in the vagina of the guinea pig. The significance of a strategy that bases the regulation of the permeability on tight junctions rather than on intercellular lipids is discussed.

Animals↗
PubMed1991

Permeability studies in neonatal rat lens epithelium.

Lanthanum nitrate (LN) and horseradish peroxidase (HRP) were used as tracers to study intercellular permeability as well as the existence and location of tight junctions and changes in them, if any, in the lens epithelium of Sprague Dawley rats. Thin sections of lenses taken from animals at birth and at three day intervals until the neonates were weaned at approximately 22 days were studied at the electron microscope level. At every age both tracers permeated the intercellular spaces of the anterior region epithelium and between the fiber cells of the equatorial region. The LN precipitates terminated near the apical part of the epithelium in the anterior polar region and were not seen at the epithelial-fiber interface or between fibers in this region. At the site of the tracer termination the intercellular space was found to be considerably constricted or completely sealed. The HRP precipitates, however, were present in these intercellular spaces and by using the HRP "washout" procedure could be washed out of the basolateral site of the intercellular spaces of the epithelium up to the point of constriction if a tight junction were present. The lens area occupied by epithelium with tight junctions was age related being very small in lenses from animals less than 24 hours old and becoming increasingly enlarged as the newborn aged. These findings further confirm the existence of a barrier in the form of tight junctions between epithelial cells in the central anterior region of the maturing rat lens. Moreover, our observations also provide information regarding the pattern of tight junction development and distribution during lens maturation.

Animals↗
PubMed2005

Claudins upregulation in human colorectal cancer.

In colorectal cancer tight junction molecular and morphological alterations are poorly understood. In this study, adenocarcinoma tissues and their paired normal mucosa (n = 12) were analyzed for tight junction alterations molecular. The expression of claudin-1, -3 and -4 was upregulated 5.7-, 1.5- and 2.4-fold, respectively, in colorectal tumor tissues in comparison to the normal ones. Although tight junction remains in the cancerous epithelium, its barrier function was altered. Despite claudins overexpression, paracellular permeability to ruthenium red was increased and a significant disorganization of tight junction strands was observed in freeze fracture replicas. Whereas the functional significance of claudin overexpression in colorectal cancer is unclear, these proteins can become potential markers and targets in colorectal cancer.

Adenocarcinoma↗
PubMed2006

PALS1 regulates E-cadherin trafficking in mammalian epithelial cells.

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

Adherens Junctions↗