No abstract

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.

Dietary flavonoids provide various beneficial effects for our health. We investigated the promotive effects of quercetin and myricetin on the intestinal barrier function in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER) across the monolayers increased rapidly during incubation with quercetin, peaking at 6 h. Lucifer yellow flux, a paracellular marker, was dose-dependently lower after quercetin and myricetin treatments, although quercetin exhibited a more potent effect. Immunoblot analysis of tight junction (TJ) proteins revealed that zonula occludens (ZO)-2, occludin, and claudin-1 were distributed to the actin cytoskeleton fraction by quercetin without increasing their respective whole-cell levels and this distribution was correlated with the increases in TER. The claudin-4 level was elevated by quercetin in both the cytoskeleton fraction and whole cells after 12 h. Confocal microscopy showed the assembly of claudin-1 and -4 at the TJ by quercetin. An inhibitor of protein kinase Cdelta (PKCdelta), rottlerin, enhanced the barrier function with changes in the distribution and expression of TJ proteins in a manner very similar to that of quercetin. Phosphorylation of PKCdelta indicating the enzymatic activity in the cells was decreased by quercetin after 1 h. In the kinase assay, quercetin exhibits direct inhibition of the PKCdelta isoform. This study demonstrates that quercetin enhances the intestinal barrier function through the assembly of ZO-2, occludin, and claudin-1 by inhibiting PKCdelta and the increase in claudin-4 expression has an additional role after 12 h.

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Colonic fermentation products, SCFA, have various effects on colonic functions. Here, we found that physiological concentrations of SCFA immediately promote epithelial barrier function in the large intestine. Solutions of mixed and individual SCFA were applied to the caecal walls mounted on Ussing-type chambers. Transepithelial electrical resistance (TER) increased rapidly and reached a peak 35 % higher than that in the control specimen within 10 min post application of the SCFA mixture (80 acetate, 40 propionate, 20 butyrate (mmol/l)). The Lucifer yellow permeability, a paracellular transport marker, was dose-dependently reduced by the mixed SCFA, acetate and propionate solutions. Inhibition of monocarboxylate transporter-1 did not influence the increase in TER with acetate; however, lowering the pH (from 7·5 to 5·5) clearly enhanced the effect of acetate. Non-metabolizable, bromo and chloro derivatives of SCFA also increased TER. These results suggest that passive diffusion of SCFA is dominant and the metabolism of SCFA is not required for the promotive effect of SCFA on barrier function. We also observed that individual SCFA dose-dependently increased TER in T84 and Caco-2 cells, which indicates that SCFA directly stimulate epithelial cells. Depletion of membrane cholesterol and inhibitors of phosphatidylinositol-3 kinase and Gq protein attenuated the acetate-mediated promotive effect. Finally, we found that the mucosal application of the SCFA mixture dose-dependently suppressed [ 3 H] mannitol transport from the caecal lumen to the mesenteric blood in the anaesthetized rats. We conclude that physiological concentrations of SCFA immediately enhance barrier function of the colonic epithelium through cholesterol-rich microdomain in the plasma membrane.