Increased epithelial permeability is observed in inflammatory states. However, the mechanism by which inflammatory mediators such as IFN-gamma increase epithelial permeability is unknown. We recently observed that IFN-gamma induces disassembly of tight junctions (TJ); in this study we asked whether such TJ disassembly is mediated by endocytosis of junctional proteins. The role of three major internalization pathways in disruption of TJ in IFN-gamma-treated intestinal epithelial cells was analyzed using selective inhibitors and markers of the pathways. No role for the clathrin- and caveolar-mediated endocytosis in the IFN-gamma-induced internalization of TJ proteins was observed. However, inhibitors of macropinocytosis blocked internalization of TJ proteins and junctional proteins colocalized with macropinocytosis markers, dextran and phosphatidylinositol-3,4,5-trisphosphate. Internalized TJ proteins were identified in early and recycling endosomes but not in late endosomes/lysosomes. These results for the first time suggest that IFN-gamma produces a leaky epithelial barrier by inducing macropinoytosis of TJ proteins.

Changes in epithelial tight junction protein expression and apoptosis increase epithelial permeability in inflammatory bowel diseases. The effect of the probiotic mixture VSL#3 on the epithelial barrier was studied in dextran sodium sulfate (DSS)-induced colitis in mice. Acute colitis was induced in BALB/c mice (3.5% DSS for 7 days). Mice were treated with either 15 mg VSL#3 or placebo via gastric tube once daily during induction of colitis. Inflammation was assessed by clinical and histological scores. Colonic permeability to Evans blue was measured in vivo. Tight junction protein expression and epithelial apoptotic ratio were studied by immunofluorescence and Western blot. VSL#3 treatment reduced inflammation (histological colitis scores: healthy control 0.94 +/- 0.28, DSS + placebo 14.64 +/- 2.55, DSS + VSL#3 8.43 +/- 1.82; P = 0.011). A pronounced increase in epithelial permeability in acute colitis was completely prevented by VSL#3 therapy [healthy control 0.4 +/- 0.07 (extinction/g), DSS + placebo 5.75 +/- 1.67, DSS + VSL#3 0.26 +/- 0.08; P = 0.003]. In acute colitis, decreased expression and redistribution of the tight junction proteins occludin, zonula occludens-1, and claudin-1, -3, -4, and -5 were observed, whereas VSL#3 therapy prevented these changes. VSL#3 completely prevented the increase of epithelial apoptotic ratio in acute colitis [healthy control 1.58 +/- 0.01 (apoptotic cells/1,000 epithelial cells), DSS + placebo 13.33 +/- 1.29, DSS + VSL#3 1.72 +/- 0.1; P = 0.012]. Probiotic therapy protects the epithelial barrier in acute colitis by preventing 1) decreased tight junction protein expression and 2) increased apoptotic ratio.

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane. INTRODUCTIONTwo major functions of epithelia in multicellular organisms are protection from the external environment and preservation of the unique biochemical composition within different body compartments (Schock and Perrimon, 2002). These functions require restriction of the passage of solutes and macromolecules (Madara, 1998) that depends on three types of specialized plasma membrane structures termed tight junction (TJ), adherens junction (AJ), and desmosomes (Tsukita et al., 2001;Matter and Balda, 2003). TJs and AJs are closely positioned at the apical-most aspect of the lateral plasma membrane and are functionally coupled in the regulation of paracellular permeability. Thus, the TJ and AJ are collectively referred to as the apical junctional complex (AJC). Both TJs and AJs represent multiprotein complexes consisting of integral membrane proteins and peripheral proteins associated with the cytosolic side of the plasma membrane (Yap et al., 1997;Tsukita et al., 2001;Pokutta and Weis, 2002;Matter and Balda, 2003). It is generally thought that integral membrane proteins of TJs and AJs interact with partners on the opposing cell plasma membrane in a trans manner providing mechanical forces for cell-cell adhesion and creating a physical barrier that limits diffusion of solutes and macromolecules. The transmembrane proteins of TJs include occludin, members of the claudin protein family and two immunoglobulin (Ig)-like proteins, junctional adhesion molecule (JAM)-A and coxsackie adenovirus receptor (Tsukita et al., 2001;Gonzalez-Mariscal et al., 2003;Matter and Balda, 2003). The major integral membrane component...