Initiation of apoptosis by actin cytoskeletal derangement in human airway epithelial cells

Changes in epithelial cell shape can lead to cell death and detachment. Actin filaments are cleaved during apoptosis, but whether disruption in the actin cytoskeletal network, as one manifestation of cell shape change, can itself induce apoptosis is not known. We tested this hypothesis in the airway epithelial cell line 1HAEo(-) and in primary airway epithelial cells by preventing actin filament elongation with cytochalasin D or by aggregating actin filaments with jasplakinolide. Disruption of actin filament integrity promptly induced apoptosis in adherent epithelial cells within 5 h. Jasplakinolide-induced apoptosis did not disrupt focal adhesions, whereas cytochalasin D-induced apoptosis decreased focal adhesion protein expression and occurred despite ligation of the fibronectin receptor. Death induction was abrogated by the caspase inhibitors z-VAD-fmk and Ac-DEVD-cho but not by blocking the Fas (CD95) receptor. Whereas cytochalasin D--induced apoptosis was associated with cleavage of pro-caspase-8, jasplakinolide-induced apoptosis was not. Both agents induced formation of a death-inducing signaling complex. These data demonstrate that disruption of actin filament integrity with either cytochalasin D or jasplakinolide induces apoptosis in airway epithelial cells but by different mechanisms, and suggest that actin may be an early modulator of apoptotic commitment.     

Vinblastine-induced autophagocytosis: the effect of disorganization of microfilaments by cytochalasin B

The effects of disorganization of cellular microfilaments by cytochalasin B on vinblastine-induced autophagocytosis was studied in Ehrlich ascites tumor cells in vitro. Incubation with vinblastine induced a formation of autophagic vacuoles in the cytoplasm. The disorganization of microfilaments by cytochalasin B failed to inhibit vinblastine-induced autophagocytosis. Incubation with cytochalasin B alone induced a rapid formation of blebs on the cell surface. These contained cytoplasmic organelles and were connected by a narrow shaft to the main part of the cell. Thin subcortical microfilaments seen in the control cell cytoplasm were apparently relocated after cytochalasin B treatment and formed amorphous masses deeper in the cytoplasm. Vinblastine did not affect the formation of blebs after cytochalasin B treatment.     

Pancreatic acinar cell changes induced by caerulein, vinblastine, deuterium oxide, and cytochalasin B in vitro

The effects of caerulein, vinblastine (VB), deuterium oxide (D2O), and cytochalasin B upon both the structure and the function of the pancreatic acinar cell were studied in vitro using rat pancreatic fragments. Caerulein (10 ng. per ml.) stimulates the release of enzymes and induces the appearance of numerous exocytotic images at the apical part of the acinar cell. Whereas VB (5.10(-5) M) and D2O (55 per cent) inhibit the secretory response to caerulein, they do not affect the general ultrastructure of the acinar cell. Prolonged incubation in the presence of VB provoked the disappearance of microtubles and the massive precipiation of microcrystalline material in all parts of the cytoplasmic space. Numerous microtubules were found in the acinar cell after exposure to D2O. Although VB and D2O do not alter the microfilamentous network localized at the apical part of the cell, cytochalasin B (2.10(-5) M) disrupts it. This drug decreases the number of microvilli projecting into the enlarged acinar lumen. Whether cytochalasin B is used alone or in association with VB or D2O, it inhibits the secretory response to caerulein and prevents the process of exocytosis. Thus, it is suggested that microfilaments act on a later step of the secretory cycle, i.e., exocytosis, than microtubules. The probable site of action of these latter organelles in the migration of zymogen granules toward the acinar lumen is discussed.     

Attenuation of ozone-induced airway permeability in rats by pretreatment with cyclophosphamide, FPL 55712, and indomethacin.

Exposure of rats to ozone (O3) produces an increase in airway permeability and a concomitant influx of polymorphonuclear leukocytes in the lung. These observations raise the possibility that the inflammatory cells play a role in the cellular injury and increased airway permeability after O3 exposure. This study was therefore designed to determine if the inflammatory cells or their products are essential for the O3 effect. In a series of experiments, rats were rendered leukopenic with cyclophosphamide, treated with leukotriene B4 (LTB4), or with the inhibitors of lipoxygenase or cyclooxygenase products of arachidonic acid, followed by exposure to O3. A 2-h exposure to 0.8 ppm O3 caused a significant increase in the flux of proteins and albumin in bronchoalveolar lavage (BAL) and elevated the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood. The treatment with cyclophosphamide caused a significant reduction in the circulating and pulmonary leukocytes and prevented an increase in tracheal mucosal permeability to 99mTc-DTPA and the protein and albumin flux in BAL. While the intratracheal instillation of LTB4 did not affect the permeability, tracheal permeability and albumin levels in BAL in rats treated with LTD4 antagonist FPL 55712 and exposed to O3 were lower than in the untreated O3-exposed rats. Pretreatment with indomethacin also prevented the O3 effects, as reflected by the decreased protein and albumin flux in BAL and 99mTc-DTPA transport from trachea to blood. These data show a reduction in the effect of O3 by agents that affect leukocytes or their products. The results support a mechanism of increased permeability that is dependent upon inflammatory cells and their products.     

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils.

Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils.     

Cytoskeletal role in differential adhesion patterns of normal fibroblasts and breast cancer cells inside silicon microenvironments

In this paper we studied differential adhesion of normal human fibroblast cells and human breast cancer cells to three dimensional (3-D) isotropic silicon microstructures and investigated whether cell cytoskeleton in healthy and diseased state results in differential adhesion. The 3-D silicon microstructures were formed by a single-mask single-isotropic-etch process. The interaction of these two cell lines with the presented microstructures was studied under static cell culture conditions. The results show that there is not a significant elongation of both cell types attached inside etched microstructures compared to flat surfaces. With respect to adhesion, the cancer cells adopt the curved shape of 3-D microenvironments while fibroblasts stretch to avoid the curved sidewalls. Treatment of fibroblast cells with cytochalasin D changed their adhesion, spreading and morphology and caused them act similar to cancer cells inside the 3-D microstructures. Statistical analysis confirmed that there is a significant alteration (P < 0.001) in fibroblast cell morphology and adhesion property after adding cytochalasin D. Adding cytochalasin D to cancer cells made these cells more rounded while there was not a significant alteration in their adhesion properties. The distinct geometry-dependent cell–surface interactions of fibroblasts and breast cancer cells are attributed to their different cytoskeletal structure; fibroblasts have an organized cytoskeletal structure and less deformable while cancer cells deform easily due to their impaired cytoskeleton. These 3-D silicon microstructures can be used as a tool to investigate cellular activities in a 3-D architecture and compare cytoskeletal properties of various cell lines.

Rho kinase inhibition initiates apoptosis in human airway epithelial cells

Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. The actin cytoskeleton is regulated in a hierarchical manner by Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation of stress fibers and focal adhesions. Disruption of the actin cytoskeleton causes apoptosis in airway epithelial cells. To examine further the relation of cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase would elicit apoptosis in airway epithelial cells. Inhibition with either Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and apoptosis in airway epithelial cells that was blocked by inhibiting caspase function or by inhibiting protein synthesis. Cells overexpressing constitutively active Rho kinase, but not native Rho kinase, were resistant to Rho kinase inhibitor-induced stress fiber disruption and apoptosis. Inhibition of Rho kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells. We demonstrate that Rho kinase inhibition induces airway epithelial cell apoptosis associated with changes in actin filament integrity. Our data suggest that Rho kinase may be a regulator of early initiation of apoptosis.     

Effect of agents which rearrange the cytoskeleton in vitro on the structure and function of hepatocytic canaliculi

The integrity of the cytoskeletal structure of the bile canaliculus (BC) may be necessary for bile secretion. This includes actin filaments in microvilli, cytokeratins in the pericanalicular sheath and microtubules in the surrounding cytoplasm. We studied these cytoskeletal structures and also the secretory function of the hepatocytes in tissue culture using double-label fluorescent staining and the transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase. The hepatocytes were obtained from 14-day-old male rats. They were cultured in serum-free Williams's E medium, with insulin and dexamethasone added to induce differentiation. Four treatment groups of hepatocytes were studied: (a) colchicine (10(-4)M for 1 hour), (b) cytochalasin B (4 micrograms/ml for 1 hour), (c) ethanol (30 to 90 mM for 24 hours) and (d) controls. Colchicine caused the disappearance of the microtubules and completely inhibited the secretion of fluorescein diacetate and horseradish peroxidase into the BC. It did not affect the uptake or transport of fluorescein diacetate on horseradish peroxidase to the BC. Cytochalasin B disrupted the actin filaments and caused their aggregation around the BC. The canaliculi were dilated and the microvilli were decreased but the secretion was normal. Ethanol did not affect either the structure of the cytoskeleton or inhibit secretion. It is concluded that the secretory function of the BC requires the integrity of microtubules in cultured hepatocytes. The integrity of actin is not necessary for uptake, transport, or secretion as long as the pericanalicular sheath remains intact. Ethanol had no effect on the structure or the function of the cytoskeleton of the bile secretory apparatus.     

The effects of antimitotic and microfilament disrupting agents on functions of isolated guinea-pig parietal cells

In isolated guinea-pig parietal cells pretreated for 60 min with the H₂-antagonist ranitidine, the antimitotic agents colchicine and vinblastine, the microfilament-disrupting agent cytochalasin B resulted in a concentration-dependent inhibition of histamine-stimulated acid secretion up to 80%. Ranitidine reduced histamine binding to the membrane located H₂-receptor. The anti-cytoskeletal agents inhibited the cellular histamine uptake but did not effect the histamine methyltransferase activity which was significantly reduced by ranitidine. The data suggest that cytoskeletal elements like microtubules and microfilaments are of very specific functional significance not only in the secretory process of the parietal cell but also for cellular transport mechanisms.     

Cytochalasin D accelerates the release of Newcastle disease virus from infected cells

The role of the cellular cytoskeleton in Newcastle disease virus (NDV) infection was explored in two ways. First, the extent of the association of viral proteins with the cytoskeletal fraction of chicken embryo cells was determined. NDV-infected cells, pulse-labelled with [35S]methionine with or without a subsequent chase, were fractionated into Triton X-100-soluble and cytoskeletal fractions. All NDV proteins become associated with the cytoskeletal fraction of cells subsequent to their synthesis. Mixing experiments provided evidence against nonspecific sticking of proteins with this cell fraction. Second, the functional significance of the cytoskeletal association was explored using the inhibitor cytochalasin D. In the presence of this inhibitor, the rate of release of radioactively labelled virions was accelerated 2.5-fold. Colchicine did not significantly alter the rate of virion release. Virus particles released from cytochalasin D-treated cells had the same density as virions released from untreated cells, but were slightly less infectious and contained less actin. These results suggest that functional microfilaments do not play an obligatory role in viral morphogenesis but rather function to slow virus particle release.     

Metabolic requirements for phorbol ester-induced cytoskeletal changes in renal epithelium

Alteration of the cytoskeleton and cell–substratum adhesion are important in the progression of renal carcinoma. We have previously shown that treatment of normal human renal epithelium with phorbol esters mimics the changes seen in renal carcinoma cells. In this study we have demonstrated that the phorbol ester-induced cytoskeletal reorganization is inhibited in the presence of deoxyglucose but not by cycloheximide. We have also shown that treatment of cells with cytochalasin B results in the formation of immature stress fibres restricted to the cell–substratum contact regions. These results suggest that the actin filaments elongate from the focal contacts and that structural rearrangement caused by phorbol esters is an energy-dependent phenomenon but is independent of de novo protein synthesis.     

Inhibition of Cytoskeletal Assembly by Cytochalasin B Prevents Signaling Through Tyrosine Phosphorylation and Secretion Triggered by Collagen but Not by Thrombin

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.     

The ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro.

Inflammatory cells are recruited to the parenchyma of the lung in a range of conditions where they are considered to have the ability to exert damaging effects on elements of the alveolus. The injurious effects of rat bronchoalveolar-derived inflammatory cells on an alveolar Type II epithelial cell line were therefore assessed. Inflammatory populations produced by intratracheal injection of Corynebacterium parvum or quartz caused non-lethal detachment injury to the epithelial cells on co-culture whereas control bronchoalveolar cells had no effect on epithelial cells. The pathogenic mineral dusts quartz and chrysotile asbestos caused increased detachment injury when added to co-cultures of epithelial cells and bronchoalveolar leucocyte populations; neither titanium dioxide, a control mineral dust, nor zymosan were active in this respect. Detachment injury was particularly marked when quartz was added to co-cultures of epithelial cells and inflammatory bronchoalveolar cells from quartz treated lung. On the basis of anti-protease and anti-oxidant studies, the detachment injury was found to be mediated by protease alone in the case of quartz cells and protease plus oxidant in the case of C. parvum cells. The two inflammatory bronchoalveolar cell populations were found to have increased proteolytic activity, compared to control bronchoalveolar cells, as shown by increased ability to degrade fibronectin, laminin and denatured collagen. Inflammatory bronchoalveolar cells therefore have the potential to attack elements of the septal extracellular matrix as well as to compromise the integrity of the alveolar epithelium.     

The ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro

Inflammatory cells are recruited to the parenchyma of the lung in a range of conditions where they are considered to have the ability to exert damaging effects on elements of the alveolus. The injurious effects of rat bronchoalveolar-derived inflammatory cells on an alveolar Type II epithelial cell line were therefore assessed. Inflammatory populations produced by intratracheal injection of Corynebacterium parvum or quartz caused non-lethal detachment injury to the epithelial cells on co-culture whereas control bronchoalveolar cells had no effect on epithelial cells. The pathogenic mineral dusts quartz and chrysotile asbestos caused increased detachment injury when added to co-cultures of epithelial cells and bronchoalveolar leucocyte populations; neither titanium dioxide, a control mineral dust, nor zymosan were active in this respect. Detachment injury was particularly marked when quartz was added to co-cultures of epithelial cells and inflammatory bronchoalveolar cells from quartz treated lung. On the basis of anti-protease and anti-oxidant studies, the detachment injury was found to be mediated by protease alone in the case of quartz cells and protease plus oxidant in the case of C. parvum cells. The two inflammatory bronchoalveolar cell populations were found to have increased proteolytic activity, compared to control bronchoalveolar cells, as shown by increased ability to degrade fibronectin, laminin and denatured collagen. Inflammatory bronchoalveolar cells therefore have the potential to attack elements of the septal extracellular matrix as well as to compromise the integrity of the alveolar epithelium.     

Differentiation of epithelial cells on microporous membranes

Summary Microporous membranes support the differentiation of a variety of cell types including the Madin-Darby canine kidney (MDCK) cell line and normal human epidermal keratinocytes (NHEK). Coincident with culturing these cells on microporous membranes is a change in the cytoskeletal architecture of the cells to a more in vivo-like morphology and the secretion of a basal lamina-like structure, the latter not being apparent when the cells are grown in conventional cell culture plates. The potential regulatory role of the cytoskeleton in basal lamina formation was investigated using both transmission electron microscopy and immunoprecipitation of the extracellular matrix component, laminin. Disruption of the MDCK microtubular network with nocodazole caused the early formation of an electron-dense structure resembling a basal lamina. Disruption of the microtubular network of both MDCK and NHEK cells caused an increase in synthesis or retention of beta laminin or both. Disruption of the MDCK microfilament network with cytochalasin D also increased the synthesis or retention of beta laminin or both, but in addition resulted in different laminin-immunoreactive proteins secreted from the cells. Thus both ultrastructural evidence and biochemical data suggest that altering the cytoskeleton of epithelial cells might affect basal lamina formation. Our data suggest that the cytoskeleton be considered one of the possible regulators of basal lamina deposition.     

Mechanosensitivity of nicotinic receptors

Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased??without change in current amplitude??when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with M??CD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn??s AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR??s mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.     

The Cytoskeleton of uterine epithelial cells: a new player in uterine receptivity and the plasma membrane transformationo

During early pregnancy and the period of blastocyst attachment,the plasma membrane of uterine epithelial cells, which is thefirst site of contact between maternal and fetal cells, undergoesa remarkable change in configuration, with morphological andbiochemical alterations occurring apically and basolaterally.These alterations are collectively referred to as ‘theplasma membrane transformation’ of early pregnancy. Itwould be remarkable if this transformation did not also involvealterations in cytoskeletal elements and, in particular, themembrane-associated cytoskeleton. This review therefore, afteran overview of the morphological and molecular aspects of themembrane transformation as background, proceeds to examine whatis known about the cytoskeleton of uterine epithelial cells.Cytoskeletal elements particularly associated with the plasmamembrane are then examined and some new approaches to understandingmembrane-skeletal dynamics, including detergent-permeabilizationtechniques for transmission and high-resolution scanning electronmicroscopy of uterine epithelial cells, are reported, togetherwith recent work on these structures. The review concludes withan examination of how membrane-skeletal elements could contributeto the membrane transformation.     

Shiga Toxin-Producing Escherichia coli Can Impair T84 Cell Structure and Function without Inducing Attaching/Effacing Lesions

Enteropathogenic Escherichia coli (EPEC) intimately adhere to epithelial cells producing cytoskeletal rearrangement with typical attaching and effacing lesions and altered epithelial barrier and transport function. Since EPEC and Shiga toxin-producing E. coli (STEC) share similar genes in the "locus for enterocyte effacement" (LEE) thought to cause these changes, it has been assumed that STEC shares similar pathogenic mechanisms with EPEC. The aims of this study were to compare the effects of EPEC and STEC on bacterial-epithelial interactions and to examine changes in epithelial function. T84 monolayers were infected with STEC O157:H7 (wild strain EDL 933 or non-toxin-producing strain 85/170), EPEC (strain E2348/69), or HB101 (nonpathogenic) and studied at various times after infection. EPEC bound more avidly than EDL 933, but both strains exhibited greater binding than HB101. Attaching and effacing lesions and severe disruption to the actin cytoskeleton were observed in EPEC by 3 h postinfection but not in EDL 933 or HB101 at any time point. EPEC and EDL 933 increased monolayer permeability to [(3)H]mannitol 5- to 10-fold. In contrast to EPEC, EDL 933 completely abolished secretagogue-stimulated anion secretion as assessed under voltage clamp conditions in Ussing chambers. Several other STEC strains induced changes similar to those of EDL 933. In conclusion, STEC impairs epithelial barrier function and ion transport without causing major disruption to the actin cytoskeleton. Pathogenic factors other than products of LEE may be operant in STEC.     

Cytoskeletal Regulation of Epithelial Barrier Function During Inflammation

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.The epithelium plays an important role in inflammation by serving as an interface between invading pathogens and the immune system of the host. Under physiological conditions, polarized epithelia form a protective barrier that allows regulated paracellular fluxes of solutes and nutrients as well as antigen sampling and surveillance by mucosal immune cells. However, during inflammation, this protective mechanism becomes compromised by various stimuli that originate on both sides of the epithelial barrier. On the apical (luminal) side, invading pathogenic microorganisms increase epithelial permeability to gain access into host tissue. The pathogens release a variety of epithelial barrier-disrupting agents that include pore-forming toxins, cytoskeleton-modifying proteins, and bacterial lipopolysaccharide (LPS). On the basal (tissue) side of the epithelial layer, activated immune cells also induce barrier disruption to facilitate their movement to sites of pathogen invasion. Mucosal immune cells increase epithelial permeability by secreting proinflammatory cytokines such as interferon (IFN) γ, tumor necrosis (TNF) α, and interleukin (IL)−1β or by releasing proteases and reactive oxygen species (ROS). As a result, mucosal inflammation commonly leads to sustained epithelial barrier compromise, which increases body exposure to external noxious agents, thereby further exaggerating the inflammatory response. Consequently it is believed that decreasing epithelial permeability may have beneficial effects by limiting inflammatory responses. Thus, understanding mechanisms that control the epithelial barrier disruption is important in identifying novel molecular targets for pharmacological modulation of mucosal inflammation.Properties of the epithelial barrier are regulated by specialized plasma membrane structures referred to as apical junctions. These structures are composed of adhesive and scaffolding proteins that are anchored into different cytoskeletal structures such as actin filaments, intermediate filaments, and microtubules. During inflammation, it is known that reorganizations of apical junctions mediate epithelial barrier dysfunction. Mounting evidence suggests that the actin cytoskeleton plays a pivotal role in regulating junctional integrity and remodeling under physiological and pathological states.In this review, we will discuss the role of actin filaments in the regulation of epithelial barrier integrity and its breakdown during inflammation. How inflammatory processes in different mucosal tissues induce remodeling of junction-associated filaments (F) actin and alter structure and barrier properties of epithelial cell-cell adhesions will be discussed. While epithelial junctions will be the major focus of this review, we will occasionally refer to examples of junctional regulation in the vascular endothelium. Likewise, we limit the discussion to simple columnar epithelia and exclude complex stratified epithelia such as the epidermis. Finally, we specifically focus on the role of actin filaments in maintenance and disassembly of epithelial junctions without discussing junctional reassembly during epithelial wound healing.     

Cortactin deficiency causes increased RhoA/ROCK1-dependent actomyosin contractility,intestinal epithelial barrier dysfunction,and disproportionately severe DSS-induced colitis

The intestinal epithelium constitutes a first line of defense of the innate immune system. Epithelial dysfunction is a hallmark of intestinal disorders such as inflammatory bowel diseases (IBDs). The actin cytoskeleton controls epithelial barrier integrity but the function of actin regulators such as cortactin is poorly understood. Given that cortactin controls endothelial permeability, we hypothesized that cortactin is also important for epithelial barrier regulation. We found increased permeability in the colon of cortactin-KO mice that was accompanied by reduced levels of ZO-1, claudin-1, and E-cadherin. By contrast, claudin-2 was upregulated. Cortactin deficiency increased RhoA/ROCK1-dependent actomyosin contractility, and inhibition of ROCK1 rescued the barrier defect. Interestingly, cortactin deficiency caused increased epithelial proliferation without affecting apoptosis. KO mice did not develop spontaneous colitis, but were more susceptible to dextran sulfate sodium colitis and showed severe colon tissue damage and edema formation. KO mice with colitis displayed strong mucus deposition and goblet cell depletion. In healthy human colon tissues, cortactin co-localized with ZO-1 at epithelial cell contacts. In IBDs patients, we observed decreased cortactin levels and loss of co-localization with ZO-1. Thus, cortactin is a master regulator of intestinal epithelial barrier integrity in vivo and could serve as a suitable target for pharmacological intervention in IBDs.