TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Dynamics of tight and adherens junctions under EGTA treatment

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.     

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.     

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

The effects of physiologically relevant increase in temperature (37-41 degrees C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37-41 degrees C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37-41 degrees C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41 degrees C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability (P < 0.001). Heat exposure (41 degrees C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress.     

The protective effect of trefoil factor 3 on the intestinal tight junction barrier is mediated by toll-like receptor 2 via a PI3K/Akt dependent mechanism

Trefoil factor peptides are highly conserved secreted molecules characterized by heat and enzymatic digestion resistance. Intestinal trefoil factor 3 (TFF3) protects and repairs the gastrointestinal mucosa and restores normal intestinal permeability, which is dependent on the integrity of the tight junction (TJ) barrier and the TJ associated proteins claudin-1, zona occludens-1 (ZO-1) and occludin. Despite the important role of intestinal barrier dysfunction in the pathogenesis of inflammatory bowel diseases, the underlying mechanisms and associated molecules remain unclear. In the present study, we show that TFF3 and toll-like receptor 2 (TLR2) are functionally linked and modulate intestinal epithelial permeability via a mechanism that involves the PI3K/Akt pathway. We used the Caco-2 cell model to show that TLR2 and TFF3 inhibit the IL-1β induced increase in permeability and release of proinflammatory cytokines, and that this effect is mediated by activation of PI3K/Akt signaling. TLR2 silencing downregulated the expression of TFF3 and overexpression of TLR2 and TFF3 increased the levels of phospho-Akt. TFF3 overexpression significantly upregulated the expression of ZO-1, occludin and claudin-1 and this effect was abrogated by inhibition of the PI3K/Akt pathway. Taken together, our results indicate that TLR2 signaling selectively enhances intestinal TJ barrier integrity through a mechanism involving TFF3 and the activation of the PI3K/Akt pathway.     

Dynamics of enterocyte tight junctions: effect of experimental colitis and two different anti-TNF strategies

An alteration of the intestinal barrier is considered to represent an early step in pathogenesis of Crohn's disease. The integrity of intestinal barrier function is guaranteed among other factors by enterocyte tight junction (TJ) proteins. Clinical and experimental data indicate the TNF-alpha to be the major responsible factor for these defects. In the present study we investigated the very early effects of DNBS-ethanol colitis on ileal enterocyte TJ proteins [occludin, zonula occludens-1 (ZO-1), claudin-2] in controls, mice treated with infliximab (IFX) or with etanercept (ETC), and in knockout mice for the TNF-alpha receptor 1 (TNFR-1(-/-)). Circulating TNF-alpha levels were effectively reduced by IFX and ETC (P < 0.01, both) at 3 and at 6 h. DNBS colitis induced disappearance of occludin and ZO-1 from enterocyte cell-cell contact, whereas claudin-2, absent under control conditions, appeared in the ileal epithelium. These alterations were prevented equally by both treatments, IFX and ETC, and in TNFR-1(-/-) animals. DNBS colitis induced a very rapid loss of occludin and ZO-1 from ileal TJ together with an upregulation of claudin-2. Our data are consistent with the hypothesis that TNF-alpha is involved in early TJ rearrangement and that its effects are mediated through TNFR-1. Despite clinical differences, both anti-TNF treatments were equally effective in the present setting.     

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption

Oxidants such as monochloramine (NH(2)Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH(2)Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH(2)Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH(2)Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH(2)Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH(2)Cl. The actin-stabilizing agent phalloidin did not prevent NH(2)Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH(2)Cl, observed as a redistribution of the alpha-subunit of basolateral Na(+)-K(+)-ATPase to the apical membrane, an effect not found with the apical membrane protein Na(+)/H(+) exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.     

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.     

Kidney claudin-19: localization in distal tubules and collecting ducts and dysregulation in polycystic renal disease

Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin-19, in kidneys. Mouse claudin-19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary-related claudins are claudin-1 and -7, which share approximately 75% DNA sequence homology with claudin-19. Claudin-19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT-PCR. Claudin-19 and zonula occludens-1 (ZO-1) are localized at junctional regions of Madin-Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO-1 is found in the claudin-19-associated protein complexes in MDCK cells by co-immunoprecipitation. Using aquaporin-1 and aquaporin-2 antibodies as markers for different renal segment, strong expression of claudin-19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin-19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin-19 immunoreactivity is found co-localized with the ZO-1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin-19 and ZO-1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin-19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin-19 are noticed, suggesting a possible correlation between claudin-19 and renal disorders. Taken together, claudin-19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.     

Organization and formation of the tight junction system in human epidermis and cultured keratinocytes

Occludin and several proteins of the claudin family have been identiried in simple epithelia and in endothelia as major and structure-determining transmembrane proteins clustered in the barrier-forming tight junctions (TJ), where they are associated with a variety of TJ plaque proteins, including protein ZO-1. To examine whether TJ also occur in the squamous stratified epithelium of the interfollicular human epidermis we have applied several microscopic and biochemical techniques. Using RT-PCR techniques, we have identiried mRNAs encoding protein ZO-1, occludin and claudins 1, 4, 7, 8, 11, 12, and 17 in both tissues, skin and cultured keratinocytes, whereas claudins i and 10 have only been detected in skin tissue. By immunocytochemistry we have localized claudin-1, occludin and protein ZO-1 in distinct plasma membrane structures representing cell-cell attachment zones. While claudin-1 occurs in plasma membranes of all living cell layers, protein ZO-1 is concentrated in or even restricted to the uppermost layers, and occludin is often detected only in the stratum granulosum. Using electron microscopy, typical TJ structures ("kissing points") as well as some other apparently related junctional structures have been detected in the stratum granulosum, interspersed between desmosomes. Modes and patterns of TJ formation have also been studied in experimental model systems, e.g., during wound healing and stratification as well as in keratinocyte cultures during Ca2+-induced stratification. We conclude that the epidermis contains in the stratum granulosum a continuous zonula occludens-equivalent structure with typical TJ morphology and molecular composition, characterized by colocalization of occludin, claudins and TJ plaque proteins. In addition, cell-cell contact structures and certain TJ proteins can also be detected in other epidermal cell layers in specific cell contacts. The pattern of formation and possible functions of epidermal TJ and related structures are discussed.     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

Effect of n-3 polyunsaturated fatty acids on membrane microdomain localization of tight junction proteins in experimental colitis

Ulcerative colitis (UC) is a gastrointestinal disorder characterized by an inflammatory process associated with mucosal damage. Many studies have shown that n-3 polyunsaturated fatty acids (PUFAs) possess anti-inflammatory effects in inflammatory bowel disease. The aim of this study was to investigate whether n-3 PUFAs could alleviate intestinal damage in experimental UC. In the present study, we found that in 2,4,6-trinitrobenzenesulfonic acid-induced colitic rats, the damage to the intestinal mucosa was accompanied by a disrupted tight junction (TJ) structure. In accordance with these changes, the distribution and expression of TJ proteins, including occludin, claudin-1, claudin-3, claudin-5, claudin-8 and ZO-1, in membrane microdomains was altered. The distribution of flotillin-1, a lipid raft marker protein, was also changed. Moreover, we found for the first time that n-3 PUFAs prevented redistribution of TJ proteins from Triton X-100-insoluble raft-like membrane microdomains to Triton X-100-soluble fractions. The expression of ZO-1, claudin-1, claudin-5 and claudin-8 was significantly elevated by n-3 PUFAs. n-3 PUFAs also attenuated the disruption of TJ structure and improved the histological score. Our results demonstrate that the expression and distribution of TJ proteins in TJ membrane microdomains might be affected in UC, and that such altered expression of TJ proteins in membrane microdomains in experimental UC is affected by n-3 PUFAs. These findings may have therapeutic potential in intestinal inflammation.     

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.     

Formation and barrier function of tight junctions in human ovarian surface epithelium

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.