The Reversible Increase in Tight Junction Permeability Induced by Capsaicin Is Mediated via Cofilin-Actin Cytoskeletal Dynamics and Decreased Level of Occludin

<div><p>Previous results demonstrated that capsaicin induces the reversible tight junctions (TJ) opening via cofilin activation. The present study investigated the mechanisms underlying the reversible TJ opening and compared the effect to the irreversible opening induced by actin inhibitors. Capsaicin treatment induced the F-actin alteration unique to capsaicin compared to actin-interacting agents such as latrunculin A, which opens TJ irreversibly. Along with TJ opening, capsaicin decreased the level of F-actin at bicellular junctions but increased it at tricellular junctions accompanied with its concentration on the apical side of the lateral membrane. No change in TJ protein localization was observed upon exposure to capsaicin, but the amount of occludin was decreased significantly. In addition, cosedimentation analyses suggested a decrease in the interactions forming TJ, thereby weakening TJ tightness. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079954#s1" target="_blank">Introduction</a> of cofilin, LIMK and occludin into the cell monolayers confirmed their contribution to the transepithelial electrical resistance decrease. Finally, exposure of monolayers to capsaicin augmented the paracellular passage of both charged and uncharged compounds, as well as of insulin, indicating that capsaicin can be employed to modulate epithelial permeability. Our results demonstrate that capsaicin induces TJ opening through a unique mechanism, and suggest that it is a new type of paracellular permeability enhancer.</p></div

Capsaicin decreases occludin protein content and the protein-protein interactions involving occludin in TJ.

<p>(A,B) MDCK monolayers were exposed to ethanol control or 300 µM capsaicin for 45 min. (A) The cellular localization of the TJ proteins occludin and claudin-1 was examined by immunofluorescence. Scale bar: 10 µm. (B) Western blot detection of occludin, tricellulin, Zo-1, claudin-1, E-cadherin and actin in the cytosol, membrane fractions and total cell extracts. The densitometic analysis of total proteins from three independent experiments performed with NIH ImageJ software. **represents p<0.01 by Student’s <i>t</i> test. (C) Western blot detection of occludin, claudin-1, phospho-cofilin, cofilin and E-cadherin in total extracts exposed to 300 µM capsaicin or 0.1 µM LatA for the durations indicated. (D) Distribution of TJ proteins in sucrose gradient centrifugation of lysates from monolayers (shown in the inset) after exposure to ethanol control or 300 µM capsaicin for 1 h. Fractions were collected from the top of the gradient, separated by SDS-PAGE and subjected to immunoblotting. The apparent molecular masses of BSA (66 kDa) and myosin (200 kDa) are shown above the fraction number. Representative data from three independent experiments is shown. The densitometic analyses from three independent experiments are shown. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Effects of suppression of cofilin activation or overexpresssion of TJ proteins on the capsaicin-induced cofilin dephosphorylation and decrease in occludin expression.

<p>(A–C) Flag-HA tagged cofilin and LIMK, and EGFP-occludin and claudin-1 stable transfectants were established. Monolayers prepared from the transfectants and non-transfected MDCK cells were exposed to 300 µM capsaicin for the time indicated. After lysis, protein expression was analyzed by immunoblotting. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were pretreated with vehicle or 50 µM TFP for 30 min, and then exposed to 100 µM capsaicin for the time indicated. The levels of occludin and phospho-cofilin were analyzed by western blotting.</p

Capsaicin increases TJ permeability as measured by the paracellular passage of ionic and non-ionic compounds.

<p>(A,B,D) MDCK cells were seeded on transwells and grown for 3 days to develop monolayers. Paracellular fluxes of CF (A), FD4 (B), and insulin (D) were measured in the apical to basolateral direction. All measurements were in triplicates (n = 3) and representative data are shown. (C) Effects of 300 µM capsaicin, 1 mg/ml C₁₀, 0.1 µM LatA on the P_app of molecules across MDCK monolayers. P_app was calculated from the above data. <i>^a</i>Each value represents the mean ± S.D. of three independent experiments. <i>^b</i>Enhancement ratio calculated as P_app(sample)/P_app(control).</p

Cofilin activation, decrease in occludin expression and actin alteration in the recovery phase of capsaicin treatment.

<p>(A) Western blot detection of phospho-cofilin and occludin in total extracts exposed to 300 µM capsaicin for the times indicated. (B) MDCK monolayers were exposed to 300 µM capsaicin for the time indicated, and then fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm.</p

Overexpresssion of cofilin, LIMK and occludin, but not claudin-1, significantly diminishes the capsaicin-induced decrease in TER.

<p>(A,B) Transfectant monolayers were prepared in transwell inserts, exposed to 300 µM capsaicin, and subjected to TER measurements. Values represent mean ± S.D. Asterisks indicated significant difference from Vector control at the same time point; *, p<0.01; **, p<0.001. (C) TER was assessed in MDCK monolayers pretreated with vehicle control or 50 µM TFP for 30 min, and exposed to 100 µM capsaicin at time 0. Values represent mean ± S.D. Repeated measure ANOVA followed by Tukey’s multiple comparisons test; **p<0.01, control vs. capsaicin; ^##p<0.01, TFP + capsaicin vs. capsaicin. Each experiment was performed with at least two different clones and repeated at least twice. (D) MDCK monolayers were treated with vehicle or 50 µM TFP for 30 min followed by 100 µM capsaicin for 15 min, then fixed and stained with rhodamine-phalloidin to detect F-actin alteration (upper panel). To see the relative distribution of F-actin (red) in 3D, images labeled with anti-claudin-1 (green) and Hoechst (blue) are also shown below. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm.</p

Capsaicin alters the TER of epithelial monolayers in a concentration-dependent and reversible manner.

<p>(A) TER was assessed in MDCK monolayers exposed to ethanol (vehicle) control (crosses), 300 µM (triangles), 100 µM (circles) or 30 µM (squares) capsaicin. (B) After exposure to capsaicin at the concentrations and for the lengths of time indicated, monolayers in (A) were lysed and subjected to western blot analysis. (C) MDCK monolayers incubated with ethanol control (crosses), 300 µM capsaicin (squares) or 0.1 µM LatA (triangles). Values represent mean ± S.D. Asterisks indicated significant difference from control at the same time point; *, p<0.01; **, p<0.001.</p

Capsaicin induces actin depolymerization and morphological changes.

<p>(A) Monolayers were exposed to ethanol control, capsaicin (300 µM, 45 min), Jpk (2 µM, 60 min) or LatA (0.5 µM, 90 min) and lysed, and the G- and F-actin fractions were separated. Fractions were analyzed by SDS-PAGE and immunoblotting with an anti-actin antibody. (B) MDCK monolayers were exposed to ethanol control, capsaicin (300 µM, 45 min), LatA (0.1 µM, 30 min), Jpk (2 µM, 1 min) or CytoB (5 µg/ml, 15 min), and were fixed and stained with rhodamine-phalloidin to detect F-actin. Images from each z-section were deconvoluted and overlayed. Bar: 10 µm. (C) The fluorescence intensities of the images in (B) were analyzed. Bar: 10 µm. ***represents p<0.001 by Student’s <i>t</i> test.</p

Increased CD112 Expression in Methylcholanthrene-Induced Tumors in CD155-Deficient Mice

<div><p>Tumor recognition by immune effector cells is mediated by antigen receptors and a variety of adhesion and costimulatory molecules. The evidence accumulated since the identification of CD155 and CD112 as ligands for DNAM-1 in humans and mice has suggested that the interactions between DNAM-1 and its ligands play an important role in T cell– and natural killer (NK) cell–mediated recognition and lysis of tumor cells. We have previously demonstrated that methylcholanthrane (MCA) accelerates tumor development in DNAM-1–deficient mice, and the <i>Cd155</i> level on MCA-induced tumors is significantly higher in DNAM-1–deficient mice than in wild-type (WT) mice. By contrast, <i>Cd112</i> expression on the tumors is similar in WT and DNAM-1-deficient mice, suggesting that CD155 plays a major role as a DNAM-1 ligand in activation of T cells and NK cells for tumor immune surveillance. To address this hypothesis, we examined MCA-induced tumor development in CD155-deficient mice. Unexpectedly, we observed no significant difference in tumor development between WT and CD155-deficient mice. Instead, we found that <i>Cd112</i> expression was significantly higher in the MCA-induced tumors of CD155-deficient mice than in those of WT mice. We also observed higher expression of DNAM-1 and lower expression of an inhibitory receptor, TIGIT, on CD8⁺ T cells in CD155-deficient mice. These results suggest that modulation of the expression of receptors and CD112 compensates for CD155 deficiency in immune surveillance against MCA-induced tumors.</p></div

Relative cytokine mRNA levels in MCA-induced tumors.

<p>(A, B) Fibrosarcomas induced by 5 µg MCA in WT or CD155-deficient (KO) C57BL/6N (A) or BALB/c (B) mice were resected from each mouse and subjected to quantitative qRT-PCR for transcripts of the indicated cytokines. Horizontal bars represent means and error bars represent means ± SEM. <i>P</i> Values for Student’s <i>t</i> test are shown.</p