Decrease In Occludin Expression Research Articles

Occludin Is Essential to Maintain Normal Alveolar Barrier Integrity and Its Protective Role During ARDS Progression.

Acute respiratory distress syndrome (ARDS) is a severe lung condition without targeted therapy that is characterized by the disruption of epithelial and endothelial barriers. The role of the tight junction protein occludin in the pathogenesis of this disease is unknown, although it has previously been deemed redundant in some tissues. The aim of the present study is to determine whether occludin is required for lung function by controlling alveolar barrier integrity in mouse models. Immunofluorescence staining of lungs from ARDS patients revealed a significant decrease in occludin expression compared to controls. Gene delivery of shRNA against occludin in the mouse lung reduced occludin levels and induced lung injury, as assessed by wet-to-dry-ratio, histology, and cellularity and protein content of bronchial alveolar lavage fluid. Conversely, gene delivery of an occludin-expressing plasmid increased occludin expression and dampened endotoxin-induced lung injury. In primary rat alveolar epithelial cells, occludin levels were positively correlated with barrier integrity, as well as membrane localization of claudin-18, another tight junction protein. Collectively, our data demonstrate that occludin plays a significant role in alveolar barrier function and that targeting occludin may provide a new therapeutic approach for ARDS.

Dietary dehulled adlay ameliorated alcoholic liver disease progression by modulating oxidative stress, inflammation, and gut-liver axis disruption in rats

Alcohol consumption causes alcoholic liver diseases (ALD). The aim of this study investigated whether the mechanisms of the effects of dehulled adlay on ALD by using a chronic ethanol feeding plus single binge model. Rats were pair-fed with a control liquid diets (C), ethanol liquid diet (E), or ethanol + dehulled adlay diet (EA) containing equal energy and dietary fiber for 5 weeks. The results revealed that dietary dehulled adlay ameliorated ethanol-induced increases in oxidative stress and inflammation and modulating nuclear factor erythroid 2-related factor 2-inflammasome and Toll-like receptor-4 signaling pathways. Furthermore, dehulled adlay arrested the decrease in occludin expression in the ileum and mitigated changes in the diversity and composition of the gut microbiota caused by alcohol exposure, which were significantly correlated with ALD biomarkers. Our results suggest that dietary dehulled adlay prevents ALD progression possibly by reducing hepatic oxidative stress and inflammation, and modulating gut dysbiosis.

MATRIX METALLOPROTEINASE (MMP-9) INDUCED INCREASE IN INTESTINAL EPITHELIAL TIGHT JUNCTION BARRIER IS MEDIATED BY EGFR ACTIVATION

Abstract BACKGROUND The defective intestinal tight junction (TJ) barrier has a key pathogenic factor in Crohn’s disease (CD) and other inflammatory conditions of the gut. Clinical studies in CD patients have shown that a persistent increase in intestinal permeability is predictive of poor clinical outcome. Matrix metalloproteinase-9 (MMP-9) has been shown to be markedly increased in intestinal tissues of patients with CD and it has been postulated that MMP-9 plays an important role on the pathogenesis of intestinal inflammation in CD. Our recent studies suggested that MMP-9 at clinically relevant concentrations causes an increase in intestinal epithelial TJ permeability in-vitro and in-vivo by inducing intracellular signaling pathways via activating distinct basolateral membrane receptors. However, the specific protein receptors and the intracellular signaling cascades involved have not been fully identified. One of the potential protein receptors that might be a target of MMP-9 on the basolateral membrane of the intestinal epithelial cells is the epidermal growth factor receptor (EGFR), which has been shown to have opposing roles in the pathophysiology of CD. AIMS Therefore, our aims in this study were to examine the effect of MMP-9 on EGFR activation and delineate the role of EGRF in MMP-9 induced increase in intestinal epithelial TJ permeability by using Caco-2 monolayers as an in-vitro model system. RESULTS MMP-9 (400 ng/ml) caused a time-dependent increase in EGFR activation as assessed by EGFR phosphorylation, and by in-vitro kinase activity measurement of EGFR in filter-grown Caco-2 monolayers. Inhibition of EGFR by pharmacologic inhibitor, PD 153035, prevented the MMP-9 induced increase in Caco-2 TJ permeability measured by trans-epithelial resistance (TER) and mucosal-to-serosal flux of a paracellular marker dextran 10kd. MMP-9 caused a decrease in the TJ protein occludin expression. Inhibition of EGFR by PD 153035 prevented the MMP-9 induced degradation of occludin expression. In addition, the effect of EGFR activation by a known activator, NSC 228155 25, in the absence of MMP-9, caused an increase in Caco-2 TJ permeability and a decrease in occludin expression. CONCLUSION Our data showed that MMP-9 induced increase in intestinal epithelial TJ permeability is mediated in part by the activation of EGFR and the subsequent degradation of occludin expression. Identifying the specific membrane receptors that mediate the MMP-9 induced increase in intestinal TJ permeability could be important in developing future therapeutic strategies in IBD.

Catechin and Procyanidin B2 Modulate the Expression of Tight Junction Proteins but Do Not Protect from Inflammation-Induced Changes in Permeability in Human Intestinal Cell Monolayers.

The possibility of counteracting inflammation-related barrier defects with dietary compounds such as (poly)phenols has raised much interest, but information is still scarce. We have investigated here if (+)-catechin (CAT) and procyanidin B2 (PB2), two main dietary polyphenols, protect the barrier function of intestinal cells undergoing inflammatory stress. The cell model adopted consisted of co-cultured Caco-2 and HT29-MTX cells, while inflammatory conditions were mimicked through the incubation of epithelial cells with the conditioned medium of activated macrophages (MCM). The epithelial barrier function was monitored through trans-epithelial electrical resistance (TEER), and ROS production was assessed with dichlorofluorescein, while the expression of tight-junctional proteins and signal transduction pathways were evaluated with Western blot. The results indicated that MCM produced significant oxidative stress, the activation of NF-κB and MAPK pathways, a decrease in occludin and ZO-1 expression, and an increase in claudin-7 (CL-7) expression, while TEER was markedly lowered. Neither CAT nor PB2 prevented oxidative stress, transduction pathways activation, ZO-1 suppression, or TEER decrease. However, PB2 prevented the decrease in occludin expression and both polyphenols produced a huge increase in CL-7 abundance. It is concluded that, under the conditions adopted, CAT and PB2 do not prevent inflammation-dependent impairment of the epithelial barrier function of intestinal cell monolayers. However, the two compounds modify the expression of tight-junctional proteins and, in particular, markedly increase the expression of CL-7. These insights add to a better understanding of the potential biological activity of these major dietary flavan-3-ols at intestinal level.

ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1β-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.

1,25-Dihydroxyvitamin D3 prevents toluene diisocyanate-induced airway epithelial barrier disruption.

The loss of airway epithelial integrity contributes significantly to asthma pathogenesis. Evidence suggests that vitamin D plays an important role in the prevention and treatment of asthma. However, its role in airway epithelial barrier function remains uncertain. We have previously demonstrated impaired epithelial junctions in a model of toluene diisocyanate (TDI)-induced asthma. In the present study, we hypothesized that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] may prevent TDI-induced epithelial barrier disruption. Male BALB/c mice were dermally sensitized and then challenged with TDI. The mice were then administered 1,25(OH)2D3 intraperitoneally prior to challenge with TDI. For in vitro experiments, 16HBE bronchial epithelial cells were cultured and stimulated with TDI-human serum albumin (HSA). The results revealed that the mice treated with 1,25(OH)2D3 displayed decreased airway hyperresponsiveness (AHR), suppressed neutrophil and eosinophil infiltration into the airways, as well as an increased E-cadherin and zonula occludens-1 (ZO-1) expression at the cell-cell contact sites. In vitro, exposure of the cells to TDI-HSA induced a rapid decline in transepithelial electrical resistance (TER) and an increase in cell permeability, followed by a decrease in occludin expression and the redistribution of E-cadherin, accompanied by a significant upregulation in the levels of phosphorylated extracellular signal-regulated kinase (ERK)1/2. These effects were all partly reversed by treatment with either 1,25(OH)2D3 or an ERK1/2 inhibitor. In conclusion, the findings of our study demonstrate that 1,25(OH)2D3 prevents TDI-induced epithelial barrier disruption, and that the ERK1/2 pathway may play a role in this process.

Serotonergic reinforcement of intestinal barrier function is impaired in irritable bowel syndrome.

Alterations in serotonergic (5-HT) metabolism and/or intestinal integrity have been associated with irritable bowel syndrome (IBS). To assess the effects of the precursor of 5-HT, 5-hydroxytryptophan (5-HTP), on mucosal 5-HT availability and intestinal integrity, and to assess potential differences between healthy controls and IBS patients. Fifteen IBS patients and 15 healthy volunteers participated in this randomised double-blind placebo-controlled study. Intestinal integrity was assessed by dual-sugar test and by determining the mucosal expression of tight junction proteins after ingestion of an oral bolus of 100 mg 5-HTP or placebo. Mucosal serotonergic metabolism was assessed in duodenal biopsy samples. 5-HTP administration significantly increased mucosal levels of 5-HIAA, the main metabolite of 5-HT, in both healthy controls (7.1 ± 1.7 vs. 2.5 ± 0.7 pmol/mg, 5-HTP vs. placebo, P = 0.02) and IBS patients (20.0 ± 4.8 vs. 8.1 ± 1.3 pmol/mg, 5-HTP vs. placebo, P = 0.02), with the latter group showing a significantly larger increase. Lactulose/L-rhamnose ratios were significantly lower after administration of 5-HTP (P < 0.05) in healthy controls and were accompanied by redistribution of zonula occludens-1 (ZO-1), pointing to reinforcement of the barrier. In IBS, expression of the tight junction proteins was significantly lower compared to healthy controls, and 5-HTP resulted in a further decrease in occludin expression. Oral 5-HTP induced alterations in mucosal 5-HT metabolism. In healthy controls, a reinforcement of the intestinal barrier was seen whereas such reaction was absent in IBS patients. This could indicate the presence of a serotonin-mediated mechanism aimed to reinforce intestinal barrier function, which seems to dysfunction in IBS patients.

Farrerol regulates occludin expression in hydrogen peroxide-induced EA.hy926 cells by modulating ERK1/2 activity

Endothelial tight junction is a crucial intracellular junctional structure that controls paracellular permeability across vascular endothelium. Oxidative stress-mediated elevation in endothelial permeability is associated with pathogenesis of several cardiovascular diseases. In the present research, the regulation of farrerol on occludin, a transmembrane proteins associated with endothelial tight junction, was investigated in hydrogen peroxide-induced human endothelium-derived EA.hy926 cells. Western blot analysis demonstrated that H2O2 exposure caused a significant decrease in occludin expression, but had little effect on ZO-1 expression, and the decrease of occludin expression was significantly attenuated by farrerol in a dose-dependent manner. Meanwhile, immunofluorescent staining assay also demonstrated that the loss of occludin expression induced by H2O2 exposure was restored by farrerol pretreatment. Further investigations showed that farrerol prevented H2O2-induced activation of extracellular signal-regulated kinase (ERK) 1/2 in a dose-dependent manner. The use of U0126, a specific inhibitor of MEK1/2, proved that H2O2-induced decrease of occludin in EA.hy926 cells was likely associated with activation of ERK1/2, which indicated that the regulation of farrerol on occludin expression in H2O2-induced EA.hy926 cells was likely related to the modulation of ERK1/2 activation. In conclusion, the present study demonstrates for the first time that farrerol has potential effects on oxidative stress-induced endothelial tight junction disruption and suggests that farrerol is a potential candidate for the intervention of endothelial permeability-associated cardiovascular diseases.

Analysis of tight junctions in placentas affected by chorioamnionitis: in vivo and in vitro analysis

The human placenta and fetal membranes provide a barrier regulating the transfer of materials between the mother and the developing fetus throughout gestation. Chorioamnionitis is an important risk factor for preterm delivery that is associated with high perinatal morbidity and mortality. Chorioamnionitis is the term applied to infections of the placenta and membranes resulting in high concentrations of IL- 1beta, IL-6, IL-8 and TGF-beta in the amniotic fluid (D’Alquen et al., 2005). With progression of inflammation, immune cells penetrate blood vessels and infiltrate the umbilical cord, resulting in funisitis (Romero and Mazor, 1988). In normal conditions the two important physical entities in endothelial/epithelial paracellular clefts are adherens junctions and tight junctions. Tight junction governs the paracellular movement of water, solutes and immune cells, through the intercellular space creating a boundary between the apical and basolateral sides of cellular barriers (Gruenheid and Finlay, 2003). We have evaluated the localization of tight junctions studying the Zonula Occludens-1 (ZO-1) and Occludin expressions as well as the localization of adherent junctions, testing the expression of VE-cadherin and beta-catenin in placentas from normal gestations, from preterm idiopathic deliveries and from chorioamnionitis by immunohistochemistry. In addition, we have evaluated the mRNAs by real time PCR, the protein levels of these molecules by Western blot analysis in placental tissues, and to better clarify the action of some cytokines on occludin we performed in vitro analysis of HUVEC cultures. Our more striking result is the decrease of occludin expression in placentas from chorioamnionitis and an evident action of the cytokines on this molecule.

Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be an important pathogenic factor contributing to the development of intestinal inflammation. The expression of occludin is markedly decreased in intestinal permeability disorders, including in Crohn's disease, ulcerative colitis, and celiac disease, suggesting that the decrease in occludin expression may play a role in the increase in intestinal permeability. The purpose of this study was to delineate the involvement of occludin in intestinal epithelial TJ barrier by selective knock down of occludin in in vitro (filter-grown Caco-2 monolayers) and in vivo (recycling perfusion of mouse intestine) intestinal epithelial models. Our results indicated that occludin small-interfering RNA (siRNA) transfection causes an increase in transepithelial flux of various-sized probes, including urea, mannitol, inulin, and dextran, across the Caco-2 monolayers, without affecting the transepithelial resistance. The increase in relative flux rate was progressively greater for larger-sized probes, indicating that occludin depletion has the greatest effect on the flux of large macromolecules. siRNA-induced knock down of occludin in mouse intestine in vivo also caused an increase in intestinal permeability to dextran but did not affect intestinal tissue transepithelial resistance. In conclusion, these results show for the first time that occludin depletion in intestinal epithelial cells in vitro and in vivo leads to a selective or preferential increase in macromolecule flux, suggesting that occludin plays a crucial role in the maintenance of TJ barrier through the large-channel TJ pathway, the pathway responsible for the macromolecule flux.

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

Astrocytes and Neurons Express the Tight Junction-Specific Protein Occludin in Vitro

The expression of occludin, an integral plasma membrane protein specifically located at tight junctions, was studied in various epithelial and nonepithelial tissues by means of RT-PCR, Western blotting, and immunofluorescent staining. Besides detection in epithelial and endothelial tissue, expression of occludin was found in primary and secondary cultures of neurons and astrocytes. Differentiation of astrocytes in vitro led to a marked decrease in occludin expression. Extractability of occludin from plasma membranes differed considerably between epithelial and nonepithelial cells. Following treatment with Triton X-100, occludin was completely extracted from astrocytic membranes but not from membranes derived from MDCK cells, suggesting a difference in the cytoplasmic and/or plasma membrane anchoring of occludin between these cell types.