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...

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rhoassociated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells. INTRODUCTIONThe intercellular apical junctional complex (AJC) regulates epithelial barrier function, permitting the passive entry of nutrients, ions, and water, while restricting pathogen access to underlying tissue compartments. The most apical tight junction (TJ) and its subjacent adherens junction (AJ) constitute the AJC. While TJs are responsible for maintaining the seal between epithelial cells (Tsukita et al., 2001), AJs are vital for initiating and maintaining cell-cell contacts (Yap et al., 1997).Both TJs and AJs represent multiprotein complexes composed of transmembrane proteins that affiliate with cytoplasmic plaque proteins. The former proteins mediate cellcell adhesion, whereas the latter link TJs and AJs to the cytoskeleton and participate in intracellular signaling. Transmembrane proteins in TJs include occludin, claudin family of proteins, coxsackie adenovirus receptor and junctional adhesion molecule (JAM)-A, whereas cytoplasmic plaque proteins consist of a number of scaffolding and signaling molecules, such as zonula occludens (ZO) family proteins and cingulin (Tsukita et al., 2001). In AJs, the transmembrane protein E-cadherin associates with ␣-, ␤-, and p120 catenin cytoplasmic proteins (Yap et al., 1997). Several AJ and TJ cytosolic plaque proteins have been shown to directly interact with F-actin (Mege et al., 2006) and myosin (Cordenonsi et al., 1999). Such interactions are likely to mediate the attachment of the AJC to the perijunctional actomyosin bundles, and they are thought to stabilize apical junctions and regulate their dynamics (Turner, 2000).The AJC is a highly dynamic structure and may...

The apical junctional complex (AJC), encompassing the tight junction (TJ) and adherens junction (AJ) plays a vital role in regulating epithelial cell differentiation and barrier function of simple epithelia. Both AJ and TJ are comprised of multiprotein complexes consisting of transmembrane proteins, which interact with the underlying cytoskeleton via cytoplasmic scaffold proteins. These interactions are tightly controlled by a number of signaling proteins that are critical for the regulation of the AJC function. Among these signaling molecules Rho family of small GTPases have been demonstrated to regulate the AJC function in diverse physiological and pathological states. In this review we will focus on experimental data addressing the role of Rho GTPase family members, Rho, Rac and Cdc42 in the regulation of epithelial AJC, and analyze Rho GTPase-mediated signaling pathways that control maintenance, disassembly and assembly of the AJC in epithelial cells.

Background: Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.