Role of flavonoids in intestinal tight junction regulation

The gastrointestinal tract provides a physical barrier to the diffusion of foreign materials from the lumen into the circulatory system. Impairment of the intercellular tight junction (TJ) shield, which is the major determinant of intestinal barrier function, is associated with various diseases. Dietary flavonoids demonstrate various beneficial effects on our health; however, the information regarding their effects on TJ function is quite limited. To date, four flavonoids — epigallocatechin gallate (EGCG), genistein, myricetin and quercetin — have been reported to exhibit promotive and protective effects on intestinal TJ barrier functions. Genistein, a major soybean isoflavone, protects TJ barrier function against oxidative stress, acetaldehyde, enteric bacteria and inflammatory cytokines. Genistein blocks the tyrosine phosphorylation of the TJ proteins induced by oxidative stress and acetaldehyde, which results in the disassembly of the proteins from the junctional complex. Quercetin, a flavonol, enhances intestinal TJ barrier function through the assembly and expression of TJ proteins. The change in phosphorylation status is responsible for the quercetin-mediated assembly of TJ proteins. TJ protein induction has an additional role in this effect. This review presents the recent advances in our understanding of the flavonoid-mediated promotive and protective effects on intestinal TJ barrier function with a particular focus on intracellular molecular mechanisms.     

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.     

Cystine reduces tight junction permeability and intestinal inflammation induced by oxidative stress in Caco-2 cells

Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H₂O₂ for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate–dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H₂O₂-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses.

     

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BK(Ca) channels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BK(Ca) channel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BK(Ca) channel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BK(Ca) channels displayed similar unitary conductance but greater Ca(2+) sensitivity than BK(Ca) channels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BK(Ca) α-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BK(Ca) channel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.     

IL-1beta causes an increase in intestinal epithelial tight junction permeability

IL-1beta is a prototypical proinflammatory cytokine that plays a central role in the intestinal inflammation amplification cascade. Recent studies have indicated that a TNF-alpha- and IFN-gamma-induced increase in intestinal epithelial paracellular permeability may be an important mechanism contributing to intestinal inflammation. Despite its central role in promoting intestinal inflammation, the role of IL-1beta on intestinal epithelial tight junction (TJ) barrier function remains unclear. The major aims of this study were to determine the effect of IL-1beta on intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this process, using a well-established in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. IL-1beta (0-100 ng/ml) produced a concentration- and time-dependent decrease in Caco-2 transepithelial resistance. Conversely, IL-1beta caused a progressive time-dependent increase in transepithelial permeability to paracellular marker inulin. IL-1beta-induced increase in Caco-2 TJ permeability was accompanied by a rapid activation of NF-kappaB. NF-kappaB inhibitors, pyrrolidine dithiocarbamate and curcumin, prevented the IL-1beta-induced increase in Caco-2 TJ permeability. To further confirm the role of NF-kappaB in the IL-1beta-induced increase in Caco-2 TJ permeability, NF-kappaB p65 expression was silenced by small interfering RNA transfection. NF-kappaB p65 depletion completely inhibited the IL-1beta-induced increase in Caco-2 TJ permeability. IL-1beta did not induce apoptosis in the Caco-2 cell. In conclusion, our findings show for the first time that IL-1beta at physiologically relevant concentrations causes an increase in intestinal epithelial TJ permeability. The IL-1beta-induced increase in Caco-2 TJ permeability was mediated in part by the activation of NF-kappaB pathways but not apoptosis.     

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.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

Nuclear localization of the tight junction protein ZO-2 in epithelial cells

The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.     

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA-) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.     

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.     

Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction

Disruption of epithelial barrier function was identified as one of the pathologic mechanisms in inflammatory bowel diseases (IBD). Epithelial barrier consists of various intercellular junctions, in which the tight junction (TJ) is an important component. However, the regulatory mechanism of tight junction is still not clear. Here we examined the role of focal adhesion kinase (FAK) in the epithelial barrier function on Caco-2 monolayers using a specific FAK inhibitor, PF-573, 228 (PF-228). We found that the decrease of transepithelial resistance and the increase of paracellular permeability were accompanied with the inhibition of autophosphorylation of FAK by PF-228 treatment. In addition, PF-228 inhibited the FAK phosphorylation at Y576/577 on activation loop by Src, suggesting Src-dependent regulation of FAK in Caco-2 monolayers. In an ethanol-induced barrier injury model, PF-228 treatment also inhibited the recovery of transepithelial resistance as well as these phosphorylations of FAK. In a sucrose gradient ultracentrifugation, FAK co-localized with claudin-1, an element of the TJ complex, and they co-migrate after ethanol-induced barrier injury. Immunofluorescence imaging analysis revealed that PF-228 inhibited the FAK redistribution to the cell border and reassembly of TJ proteins in the recovery after ethanol-induced barrier injury. Finally, knockdown of FAK by siRNA resulted in the decrease of transepithelial resistance. These findings reveal that activation of FAK is necessary for maintaining and repairing epithelial barrier in Caco-2 cell monolayer via regulating TJ redistribution.     

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 10⁶ oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC‐dextran flux) in Caco‐2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E‐cadherin. Claudin 3, zonula occludens‐1 (ZO1) and α‐catenin were also significantly decreased, whereas claudins 1 and 2 and β‐catenin were not altered. Substantial downregulation of occludin, claudin 4, and E‐cadherin was also observed in response to CP infection ex vivo in mouse enteroid‐derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP‐infected mouse ileum and jejunum but were unaltered in Caco‐2 cells. Further, bafilomycin‐A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco‐2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection‐induced diarrhoea.     

The tight junction as a barrier to cholesterol in canine epithelial cells

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.