Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium

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.

FIGURE 1.

IL-6 increases TJ permeability to ionic solutes without any changes to dextran flux or cell viability. A, TER was measured across Caco-2 cell monolayers incubated with varying concentrations of IL-6 (0∼100 ng/ml) for 48 h. *, p < 0.05 relative to the control value. B, TER was measured across cell monolayers before incubation and 3, 6, 12, 24, 48, and 72 h after incubation with IL-6 (0, 5, and 10 ng/ml). *, p < 0.05 relative to the control value at each time point. C and D, unidirectional FITC-dextran flux (C) was evaluated across Caco-2 cell monolayers incubated with varying concentrations of IL-6 (0∼50 ng/ml) for 48 h, and cell viability was assessed by WST assay (D). E–G, NaCl dilution potentials were measured across Caco-2 cell monolayers incubated with or without 10 ng/ml IL-6 for 48 h by the basal substitution of 65 mm NaCl with 130 mm mannitol. Representative electrophysiologic measurements (D), the statistical analysis of the dilution potentials, and PNa⁺/PCl⁻ calculated from stable dilution potentials (G) are shown. *, p < 0.05 relative to the control value. Values represent the mean ± S.E. (n = 6).

FIGURE 2.

IL-6 induces claudin-2 expression in Caco-2 cells. A, whole cell extracts and detergent-insoluble fractions of Caco-2 cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) for 48 h were immunoblotted for ZO-1, ZO-2, occludin, JAM-1, claudin-1, claudin-2, claudin-3, claudin-4, and β-actin. B, whole cell extracts of Caco-2 cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) for 48 h were immunoblotted for MLCK, E-cadherin, pMLC, and total MLC. C, whole cell extracts of Caco-2 cell monolayers incubated with varying concentrations of IL-6 (0∼50 ng/ml) for 48 h were immunoblotted for claudin-2 and β-actin. Specific bands for claudin-2 were quantitated by densitometric analysis. D, whole cell extracts of Caco-2 cell monolayers before and 1, 3, 6, 12, 24, 48 h after incubation with IL-6 (10 ng/ml) were immunoblotted for claudin-2 and β-actin. Specific bands for claudin-2 were quantitated by densitometric analysis. E, Caco-2 cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) for 48 h were immunolabeled for claudin-2 and ZO-1. The fluorescent signal intensity of claudin-2 and ZO-1 in the junctional region of cells was quantified. F, claudin-2 mRNA expression was analyzed by qPCR in cell monolayers before incubation and 3, 6, 12, 24, and 48 h after incubation with IL-6 (10 ng/ml). *, p < 0.05 relative to the control value (IL-6-free or pretreatment levels). Values represent the mean ± S.E. (n = 4).

FIGURE 3.

IL-6 induced claudin-2 expression in vivo. Mice were intraperitoneally injected with IL-6 or PBS (vehicle). Colonic mucosae were immunoblotted for claudin-2 and β-actin (A), and cryosections (8 μm in thickness) of colon were triple-labeled for claudin-2 (green), actin (red), and total DNA (blue) (B). Specific bands for claudin-2 were quantitated by densitometric analysis (A). Values represent the mean ± S.E. (n = 4). *, p < 0.05 relative to the control value.

FIGURE 4.

The MEK/ERK and PI3K pathways have a role in IL-6-mediated claudin-2 expression and TJ permeability. A, TER was measured across Caco-2 cell monolayers incubated in IL-6-free medium or in medium containing 10 ng/ml IL-6 in the absence or presence of signaling inhibitors (anti-gp130; AG490, a JAK kinase inhibitor; U0126, a MEK inhibitor; LY294002, a PI3K inhibitor; PP2, a Src inhibitor; APDC, a NFκB inhibitor) for 48 h. B, whole cell extracts of Caco-2 cell monolayers incubated in IL-6-free medium or in medium containing 10 ng/ml IL-6 in the absence or presence of signaling inhibitors (anti-gp130; U0126, a MEK inhibitor; LY294002, a PI3K inhibitor; PP2, a Src inhibitor) for 48 h were immunoblotted for claudin-2 and β-actin. Specific bands for claudin-2 were quantitated by densitometric analysis. *, p < 0.05 relative to control without inhibitors. †, p < 0.05 relative to IL-6 without inhibitors. C, whole cell extracts of Caco-2 cell monolayers before incubation and 3, 10, 30, 60, and 120 min after incubation with IL-6 (10 ng/ml) were immunoblotted for pSTAT3, pERK1/2, pAkt, total ERK, and β-actin. Specific bands for pSTAT3, pERK1/2, and pAkt were quantitated by densitometric analysis. *, p < 0.05 relative to the pretreatment value. D, whole cell extracts of Caco-2 cell monolayers before incubation and 30 and 60 min after incubation with IL-6 (10 ng/ml) in the absence or presence of signaling inhibitors (anti-gp130, U0126, and LY294002) were immunoblotted for pSTAT3, pERK1/2, pAkt, total ERK, and β-actin. E, Caco-2 cells were transiently transfected with control or MEK_wt-HA plasmids, fixed, and triple-labeled for claudin-2 (green), HA-tag (red), and total DNA (blue). Areas encircled by a dotted line indicate transfected cells (MEK_wt-HA or HA-expressing cells). *, p < 0.05 relative to the control value. Values represent mean ± S.E. (n = 4).

FIGURE 5.

IL-6 induces claudin-2 expression and increases TJ permeability from basal sides. IL-6 (10 ng/ml) was applied to the apical and/or basal aspects of Caco-2 cell monolayers. After 48 h, TER across the monolayers was measured (A) and the whole cell extracts were immunoblotted for claudin-2 (B). Specific bands for claudin-2 were quantitated by densitometric analysis. *, p < 0.05 relative to the control value. Values represent the mean ± S.E. (n = 4).

FIGURE 6.

IL-6 enhances claudin-2 promoter activity in a Cdx binding site-dependent manner. A and B, claudin-2 reporter gene plasmids were transfected to Caco-2 cells. Luciferase activity was measured in the cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) for 24 and 48 h (A). Luciferase activity was measured in Caco-2 cell monolayers incubated with varying concentrations of IL-6 (0–10 ng/ml) for 48 h (B). *, p < 0.05 relative to control treatment. C, schematic drawings of transcriptional binding sites in the WT-claudin-2 promoter and deletion mutants. D, WT-claudin-2 reporter gene plasmids and the deletion mutant plasmids were transfected to Caco-2 cells. Luciferase activity was measured in the cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) for 48 h. *, p < 0.05 relative to control treatment. †, p < 0.05 relative to WT-claudin-2 with IL-6. E, WT-claudin-2 reporter gene plasmid was transfected to Caco-2 cells. Luciferase activity was measured in the cell monolayers incubated in IL-6-free medium or in medium containing 10 ng/ml IL-6 in the absence or presence of signaling inhibitors (U0126, a MEK inhibitor; LY294002, a PI3K inhibitor) for 48 h. *, p < 0.05 relative to control treatment. Values represent the mean ± S.E. (n = 4).

FIGURE 7.

IL-6 induces Cdx2 expression in Caco-2 cells. A and B, Caco-2 cell monolayers were incubated with IL-6 (10 ng/ml) for 48 h. Cdx1 and Cdx2 mRNA expression was quantified with qPCR (A), and Cdx2 protein expression quantified with immunoblotting in cell monolayers before incubation and 3, 6, 12, 24, 48 h after incubation with 10 ng/ml IL-6. Specific bands for Cdx-2 were quantitated by densitometric analysis. *, p < 0.05 relative to 0 h. C, cell monolayers incubated in the absence or presence of IL-6 (10 ng/ml) were double-labeled for Cdx2 (green) and total DNA (blue). D, whole cell extracts of Caco-2 cell monolayers incubated in IL-6-free medium or in medium containing 10 ng/ml IL-6 in the absence or presence of signaling inhibitors (U0126, a MEK inhibitor; LY294002, a PI3K inhibitor) for 48 h were immunoblotted for Cdx2. Specific bands for Cdx-2 were quantitated by densitometric analysis. *, p < 0.05 relative to control without inhibitors. †, p < 0.05 relative to IL-6 without inhibitors. Values represent the mean ± S.E. (n = 4).

FIGURE 8.

Schematic representation showing the mechanism for the IL-6-mediated increase in the TJ permeability in intestinal epithelium cells. IL-6 activates the MEK/ERK and PI3K/Akt pathways through gp130/IL-6Rα interaction, which in turn enhances Cdx2 expression. The enhanced Cdx2 expression activates the claudin-2 promoter resulting in the increase in claudin-2 expression.