It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function. Inflammation is characterized by an influx into the mucosa of immune cells that influence epithelial function by releasing proinflammatory cytokines such as IFN-γ and TNF-α. Mucosal barrier function is regulated by the epithelial apical junctional complex (AJC) consisting of the tight junction and the adherens junction. Since the AJC regulates barrier function, we analyzed the influence of IFN-γ and TNF-α on its structure/function and determined the contribution of apoptosis to this process using a model intestinal epithelial cell line, T84, and IFN-γ and TNF-α. AJC structure/function was analyzed by confocal microscopy, biochemical analysis, and physiologic measurement of epithelial gate/fence function. Apoptosis was monitored by determining cytokeratin 18 cleavage and caspase-3 activation. IFN-γ induced time-dependent disruptions in epithelial gate function that were potentiated by coincubation with TNF-α. Tight junction fence function was somewhat disrupted. Cytokine treatment was associated with internalization of AJC transmembrane proteins, junction adhesion molecule 1, occludin, and claudin-1/4 with minimal effects on the cytoplasmic plaque protein zonula occludens 1. Detergent solubility profiles of junction adhesion molecule 1 and E-cadherin and their affiliation with “raft-like” membrane microdomains were modified by these cytokines. Inhibition of cytokine-induced apoptosis did not block induced permeability defects; further emphasizing their primary influence on the epithelial AJC structure and barrier function. Our findings for the first time clearly separate the proapoptotic effects of IFN-γ and TNF-α from their abilities to disrupt barrier function.

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

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Clin. Invest. 1991. 88:1605-1612

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and ␤-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and ␣-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology. INTRODUCTIONPolarized epithelial cells create a highly efficient and selective barrier between different tissue compartments (Madara, 1998). Two junctional complexes constitute this barrier; the most apically located tight junction (TJ) and closely adjacent adherens junction (AJ; Farquhar and Palade, 1963). TJs and AJs play an important role in regulating paracellular permeability, cell differentiation, and proliferation (Gumbiner, 1996;Vleminckx and Kemler, 1999;Yeaman et al., 1999;Matter and Balda, 2003). Both junctions represent multiprotein complexes composed of transmembrane proteins and cytosolic plaque proteins (Yap et al., 1997;Tsukita et al., 2001;Gonzáles-Mariscal et al., 2003). The former proteins mediate cell-cell adhesion, whereas the latter link TJs and AJs to the cytoskeleton and participate in intracellular signaling. Transmembrane proteins in TJs include occludin, claudins, and junctional adhesion molecule (JAM), whereas its cytoplasmic plaque proteins consist of a number of scaffolding and signaling molecules such as the zonula occludens (ZO) protein family (Tsukita et al., 2001;Gonzáles-Mariscal et al., 2003). A transmembrane AJ protein, E-cadherin, is vital for initiating and maintaining cell-cell contacts (Gumbiner, 1996;Vleminckx and Kemler, 1999) and has unique calciumbinding properties (Koch et al., 1999). The cytoplasmic platform of AJs is comprised of several members of the catenin protein family interacting with E-cadherin (Yap et al., 1997).Despite complex organization, neither AJs nor TJs are static structures, and they can be rapidly disassembled and reorganized in response to various extracellular stimuli (Gumbiner, ...

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

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.

Increased epithelial permeability is a common and important consequence of mucosal inflammation that results in perturbed body homeostasis and enhanced exposure to external pathogens. The integrity and barrier properties of epithelial layers are regulated by specialized adhesive plasma membrane structures known as intercellular junctions. It is generally believed that inflammatory stimuli increase transepithelial permeability by inducing junctional disassembly. This review highlights molecular events that lead to disruption of epithelial junctions during inflammation. We specifically focus on key mechanisms of junctional regulation that are dependent on reorganization of the perijunctional F-actin cytoskeleton. We discuss critical roles of myosin-II-dependent contractility and actin filament turnover in remodeling of the F-actin cytoskeleton that drive disruption of epithelial barriers under different inflammatory conditions. Finally, we highlight signaling pathways induced by inflammatory mediators that regulate reorganization of actin filaments and junctional disassembly in mucosal epithelia.