Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the underlying interstitium. This intestinal barrier is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised in a number of intestinal diseases, including inflammatory bowel disease (IBD). In vitro studies have demonstrated that proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), that are increased in the intestinal mucosa of patients with IBD can induce a leaky mucosal barrier. There is a growing evidence that the increased permeability and altered AJC structure observed in IBD are mediated by internalization of junctional proteins. This review summarizes barrier defects observed in IBD and addresses mechanisms by which proinflammatory cytokines, such as IFN-gamma and TNF-alpha, modulate AJC structure and epithelial barrier function.

The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

The mechanisms by which enteric commensal microbiota influence maturation and repair of the epithelial barrier are relatively unknown. Epithelial restitution requires active cell migration, a process dependent on dynamic turnover of focal cell-matrix adhesions (FAs). Here, we demonstrate that natural, commensal bacteria stimulate generation of reactive oxygen species (ROS) in intestinal epithelia. Bacteria-mediated ROS generation induces oxidation of target cysteines in the redox-sensitive tyrosine phosphatases, LMW-PTP and SHP-2, which in turn results in increased phosphorylation of focal adhesion kinase (FAK), a key protein regulating the turnover of FAs. Accordingly, phosphorylation of FAK substrate proteins, focal adhesion formation, and cell migration are all significantly enhanced by bacterial contact in both in vitro and in vivo models of wound closure. These results suggest that commensal bacteria regulate cell migration via induced generation of ROS in epithelial cells.intestine | gastroenterology | phosphoprotein phosphatases | probiotics | lactobacillus T he mammalian gastrointestinal tract is home to an extraordinarily large group of commensal bacteria that mediate homeostatic effects on their host and influence a wide range of systemic metabolic, nutritional, and immune functions (1, 2). Additionally, the intestinal microbiota can directly affect the function of the epithelial cells that form a physical interface between the host and the luminal contents. For example, gut commensal bacteria have been implicated in regulation of epithelial proliferation, survival, barrier function, and resolution of epithelial wounds (3-6). In this report, we investigated the mechanisms by which the intestinal microbiota influence epithelial cell restitution.Epithelial cell restitution is a process during which wounds or breaks in the epithelial lining are repaired by migration of the surrounding epithelial cells. Cells at the leading edge flatten and move into the wounded area by rapidly extending lamellipodia, which are stabilized to the underlying matrix at specialized points called focal adhesions (FAs). The rapid disassembly of FAs at the rear end and assembly of FAs at the leading edge of the cells provides the traction force necessary for the cells to move forward (7). Additionally, FAs serve as signaling nidus points where multiple intracellular and extracellular signals integrate to coordinate cell migration. FAs are composed of protein complexes including transmembrane integrins, cytoplasmic signaling adaptors, and components of the actin cytoskeleton (8). A key regulatory protein of FA dynamics is focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that is phosphorylated in response to many extracellular signals. Models of cell migration demonstrate that phosphorylation of FAK and Src accompanies the formation of the FA complex, which subsequently mediates the turnover of adhesions and affects cell migration (7).Recently, we reported that commensal bacteria induce the generation of reactive oxygen s...