Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation

Abstract

Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.

Keywords: Autophagy; Claudin-2; Epithelial Cell; Intestinal Epithelium; Protein Degradation; Tight Junction.

FIGURE 1.

Starvation enhances the epithelial barrier function of filter-grown Caco-2 monolayers. A, filter-grown Caco-2 monolayers were incubated in starvation medium (Earle's balanced salt solution), and TER was measured over a 96-h experimental period. Starvation significantly increased TER over the normally fed control group (*, p < 0.01). B and C, starvation did not cause any change in the apical-to-basal flux of the paracellular macromolecular probes 10-kD dextran (molecular radius, 23 Å) and inulin (molecular radius, 15 Å) (flux measured after 96 h of starvation).

FIGURE 2.

Starvation reduces the TJ cation selectivity and small solute permeability of filter-grown Caco-2 monolayers. A, the NaCl dilution potential was measured after formation of an apical-to-basal electrochemical gradient, as detailed under “Experimental Procedures.” The change in dilution potential was reduced significantly after starvation (for 96 h) compared with the control group (*, p < 0.001). Similar results were obtained after the formation of a basal-to-apical electrochemical gradient (data not shown). B, starvation changed the TJ ion selectivity and reduced the ratio of the permeability of Na⁺ to Cl⁻, as calculated from the dilution potentials and the Goldman-Hodgkin-Katz equation, detailed under “Experimental Procedures” (*, p < 0.001 versus control). C—E, starvation did not affect the paracellular flux of mannitol (molecular radius, 4.1 Å) and l-glucose (molecular radius, 4.3 Å) but progressively reduced the flux of small solute urea (molecular radius, 2.9 Å) (*, p < 0.01 versus control).

FIGURE 3.

Starvation induced changes in claudins. A, Caco-2 cells were incubated in starvation medium for the indicated time and analyzed by Western blotting for claudins. β-actin is shown as a loading control. The protein levels of claudin-2, but not claudin-1, 3, 8, and 13, were reduced during starvation. B, the densitometry analysis was performed using ImageJ software to indicate the relative levels of claudin-2 after starvation. The band density in the control group was set to 1. The graph is representative of more than three independent experiments (*, p < 0.01 versus control). The starvation-induced reduction in claudin-2 protein expression correlated linearly with the increase in Caco-2 TER (C) and the reduction in paracellular flux of urea (D) (p < 0.001).

FIGURE 4.

Starvation induces autophagy in Caco-2 cell monolayers. A, Caco-2 cells were incubated in starvation medium for the indicated time and analyzed by Western blotting for LC3B and p62 protein. β-actin is shown as a loading control (Con). B, the ratio of LC3-II/LC3I density was calculated using ImageJ software. The LC3-II/LC3I ratio in the control (Cont) group was set to 1. The Western blot in A and the graph are representative of more than four independent experiments (*, p < 0.01 versus control). C, Caco-2 cells were incubated in starvation medium and bafilomycin A (20 nm) for the indicated time and analyzed by Western blotting for LC3B protein level. D, autophagic flux is represented by the accumulation of LC3II in the presence of bafilomycin A (Baf) (as shown in C) and not in the presence of bafilomycin A (as shown in A) during starvation (*, p < 0.01). E, confocal immunofluorescence for LC3 staining in starved cells showed cytoplasmic punctum formation. Scale bar = 10 μm. Shown is a representation of 48 h of starvation. F, starvation did not induce apoptosis in Caco-2 cells. Caspase-3 and caspase-7 protein expression did not change during starvation. Also, cleaved caspase-3 and -7 were not observed during starvation. Blots are representative of three independent experiments.

FIGURE 5.

Rapamycin-induced autophagy enhances TJ barrier function. A, incubation of Caco-2 cells in normal medium with the mTOR inhibitors rapamycin and PP242 (500 nm) increased TER compared with cells in starvation medium. B, Caco-2 cells were treated with rapamycin for the indicated time and analyzed by Western blotting for LC3B protein level. β-actin is shown as a loading control. The LC3 II/I densitometry ratio was found to be increased during rapamycin treatment. *, p < 0.01 versus time 0. C, the rapamycin (RAPA)-induced increase in TER was inhibited by the autophagy inhibitors bafilomycin A (BAF, 20 nm), chloroquine (CHQ, 20 μm), and wortmannin (WORT, 200 nm). *, p < 0.01 versus all other groups. D, rapamycin treatment reduced the paracellular flux of urea compared with control cells. The autophagy inhibitors bafilomycin A, chloroquine, and wortmannin attenuated the rapamycin-induced reduction in urea flux. The flux was measured after 96 h of rapamycin treatment. *, p < 0.01 versus control; #, p < 0.01 versus rapamycin. E, rapamycin treatment of Caco-2 cells in normal media led to a reduction in claudin-2 protein level. β-actin is shown as a loading control, and densitometry is represented as the claudin-2:β-actin ratio. *, p < 0.01 versus time 0.

FIGURE 6.

Autophagy inhibition attenuates the starvation-induced enhancement in the TJ barrier. A, incubation of Caco-2 cells in starvation medium with bafilomycin A (20 nm), chloroquine (CHQ, 20 μm), and wortmannin (200 nm) significantly inhibited the increase in TER caused by starvation. *, p < 0.01 versus all other groups. B, the respective siRNA transfection, but not NT siRNA transfection, produced a significant knockdown of ATG16L1 and ATG7 protein expression. C, inhibition of the autophagy-related proteins ATG16L1 and ATG7 with siRNA transfection, but not non-target siRNA transfection, significantly inhibited the increase in TER caused by starvation. * and #, p < 0.01 versus starvation. D, ATG 16L1 and ATG7 siRNA, but not non-target siRNA transfection, significantly attenuated the starvation-induced reduction in paracellular flux of urea (96 h of starvation). *, p < 0.01 versus all other groups. E and F, autophagy inhibition during starvation by bafilomycin A (BAF), chloroquine, and wortmannin (WORT) (E) and ATG16L1 and ATG7 siRNA (F) prevented a starvation-induced reduction in claudin-2 protein level. β-actin is shown as a loading control (con). The blots represent at least three independent experiments. The claudin-2:actin ratio represents the densitometry analysis. *, p < 0.01 versus all other groups.

FIGURE 7.

Overexpression of claudin-2 attenuates starvation-induced changes in TJ barrier function. A, overexpression of human CLDN2 cDNA into Caco-2 cells (Caco-2^CLDN2), as detailed under “Experimental Procedures,” led to an increase in claudin-2 protein level, as assessed by Western blot analysis. *, p < 0.01 versus control empty vector-expressing cells (Caco-2^pEZM02). B, the starvation-induced increase in TER was inhibited significantly in claudin-2 overexpressing cells (Caco-2^CLDN2). *, p < 0.01. C, starvation caused a modest reduction in urea flux in Caco-2^CLDN2 cells compared with Caco-2^pEZM02 cells. *, p < 0.01.

FIGURE 8.

Claudin-2 is targeted to lysosomes during starvation. A, confocal immunofluorescence of claudin-2 shows loss of claudin-2 (green) staining from the membrane and colocalization with the lysosomal marker LAMP2 (red; yellow in Merge panels, arrows) during starvation. The subtracted panels show only the colocalization signal. Scale bar = 2.5 μm. B, occludin (green) showed a persistent presence on the membrane and no colocalization with LAMP2 (red) during starvation. Scale bar = 5 μm.

FIGURE 9.

Increased association of claudin-2 with lysosomes during starvation. Coimmunoprecipitation studies showed an increased presence of claudin-2 in lysosomal marker LAMP2 immunoprecipitates (IP) during early starvation. The negative control (-ve con) shows immunoprecipitation with control IgG. The densitometry analysis is shown as the ratio of claudin-2:LAMP2 expression. *, p < 0.01 versus control.

FIGURE 10.

Starvation enhances the epithelial barrier function of filter-grown MDCK II monolayers. A, filter-grown MDCK II monolayers, when incubated in starvation medium, show an increase in TER over the normally fed control group (*, p < 0.01). B, starvation changed TJ ion selectivity and reduced the ratio of the permeability of Na⁺ to Cl⁻, as calculated from the dilution potentials and the Goldman-Hodgkin-Katz equation, detailed under “Experimental Procedures” (*, p < 0.001 versus control). C and D, starvation reduced the flux of small solute urea (molecular radius, 2.9 Å) (C; *, p < 0.01 versus control) but did not affect the paracellular flux of inulin (molecular radius, 15 Å, D) (flux measured after 96 h of starvation).

FIGURE 11.

Starvation induces autophagy and enhances the TJ barrier in a claudin-2-dependent manner in MDCK II cells. A, in a Western blot analysis, starvation reduced the claudin-2 protein level (representation of three blots, 96 h of starvation) (*, p < 0.01 versus control). B, confocal immunofluorescence of claudin-2 shows loss of claudin-2 (green) staining from the membrane and colocalization with the lysosomal marker LAMP2 (red, arrows). Shown is a representation of 96 h of starvation. Scale bar = 5 μm. C, confocal immunofluorescence for LC3 staining in starved MDCK II cells showed cytoplasmic punctum formation. Scale bar = 5 μm. Shown is a representation of 48 h of starvation. D, incubation of MDCK II cells in normal medium with the mTOR inhibitors rapamycin (RAPA) and PP242 (500 nm) increased the TER, comparable with cells in starvation medium. E, incubation of MDCK II cells in starvation medium with the autophagy inhibitors bafilomycin A (BAF-A), chloroquine (CHQ), and wortmannin (WORT) significantly inhibited the increase in TER caused by starvation alone. *, p < 0.01 versus all other groups.

FIGURE 12.

Claudin-2 depletion enhances TJ barrier function in Caco-2 and MDCK II cells. Claudin-2 siRNA transfection led to a significant decrease in claudin-2 protein expression in Caco-2 and MDCK II cells (A and D, respectively, Western blot). *, p < 0.01 versus NT siRNA cells. Claudin-2 siRNA transfection caused an increase in the TER of Caco-2 (B) and MDCK II (E) cells. *, p < 0.01 versus NT siRNA cells. Claudin-2 siRNA transfection also caused a reduction in urea flux in Caco-2 (C) and MDCK II cells (F). *, p < 0.01 versus NT siRNA cells. Shown are representations of 96 h post-transfection.