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Articles in PresS. Am J Physiol Gastrointest Liver Physiol (October 20, 2005). doi:10.1152/ajpgi.00301.2005 1

Roles of ZO-1, Occludin, and Actin in Oxidant-Induced Barrier Disruption Mark W. Musch*, Margaret Mary Walsh-Reitz*, and Eugene B. Chang+ The Martin Boyer Laboratories, Inflammatory Bowel Disease Research Center, Department of Medicine, The University of Chicago +

To Whom Correspondence should be addressed: Eugene B. Chang, M.D. Martin Boyer Professor of Medicine The University of Chicago Hospitals 5841 S. Maryland Avenue MC 6084 Chicago, IL 60637

Phone: 773-702-6458 fax: 773-702-2281 email: [email protected] Key words: oxidants; actin cytoskeleton; tight junctions; transepithelial electrical resistance

Acknowledgements: This work was supported by NIH grants DK-38510 and DK-47722 (EBC), Digestive Disease Core Center DK-40922, and support from the Gastrointestinal Research Foundation of Chicago.

* contributed equally

Abstract Oxidants such as monochloramine (NH2Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional

Copyright © 2005 by the American Physiological Society.



These effects can, in theory, disturb cell polarization and affect critical

membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH2Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco2 epithelial monolayers. NH2Cl causes a time-dependent decrease in both detergent-insoluble and -soluble ZO-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer, cytochalasin D, resulted in a decreased transepithelial resistance (TER) more quickly than NH2Cl, but caused a more modest and slower reduction in ZO-1 levels and in occludin re-distribution. No changes in the cellular distribution of claudin 1 or 5 or ZO-2 were observed after NH2Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH2Cl.

The actin stabilizing agent, phalloidin, did not prevent NH2Cl-

induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol.

However, it partially blocked changes in ZO-1 and

occludin distribution. Tight junctional fence function was also compromised by NH2Cl, observed as a redistribution of the alpha subunit of the basolateral Na+-K+-ATPase to the apical membrane, an effect not found with the apical membrane protein NHE3. In conclusion, oxidants not only disrupt perijunctional actin, but also cause redistribution of tight junctional proteins resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.


Introduction Barrier function is an essential task of all epithelia dependent on both the integrity and contribution of many cytoskeletal and membrane proteins that comprise and regulate tight junctional complexes that exist between cells (10, 14, 32, 39). Molecular fence function is the property of tight junctions important for maintaining segregation of membrane proteins in polarized epithelia. Barrier function is highly regulated, allowing the epithelium to control transmucosal permeability to solutes, water and electrolytes (2, 23, 30, 37). The tight junctional complex comprises a large number of membraneassociated and membrane proteins, the latter including occludin, JAM (Junction Adhesion Molecule) and the claudins (3, 6, 7, 9) that are responsible for forming the physical connections between cells that confer the basic barrier properties. Occludin has been studied extensively and has a large cytoplasmic extension that may coordinate with junctional proteins in the cytosol (3, 25, 37, 39). Occludin associates with the first tight junction-associated protein identified, ZO-1 (zonula occludens protein-1, 41).


interacts not only with occludin, but also with a number of other tight junction associated proteins including ZO-2, ZO-3, -catenin, paxillin, talin, and the perijunctional actin ring (11, 39, 44). The perijunctional actin ring is also important for maintaining barrier function, as the disruption of actin cytoskeleton by cytochalasin causes increased permeability to small solutes (20, 22, 23). Because ZO-1 serves as an important linker molecule between perijunctional actin and occludin, annular contraction in perijunctional actin can dynamically affect the intercellular permeability.


In addition to its importance regulating movement of solutes in the paracellular pathway, the tight junctions also play an important role in the maintenance of cell polarity. This fence function limits movement of proteins and lipids from the apical to basolateral (and vice versa) poles of the cells (15, 16, 24). The ability of the junctional complexes to regulate the fence function may be compromised by a number of stimuli including Ca++-dependent disruption of the tight junctions, viral or bacterial infection of cell monolayers (15, 24, 28, 29).

Although less is known about fence function as

compared to gate function, the distribution and expression of tight junctional proteins as well as the perijunctional actin may also regulate the fence function. Compromises in intestinal epithelial gate and fence functions are common in both acute and chronically inflamed mucosa. In the latter, these changes are in large part due to specific down regulation of key tight junction associated proteins such as occludin and ZO-1 by pro-inflammatory cytokines (13, 30, 42). However, in the context of acute (4 from each panel) obtained from the series of xy slices (0.2µm apart).

Arrows are indicated on

apical membranes of monolayers to indicate apical movement of alpha subunit.


images have been deconvolved using the unsharpmask and despeckle routines in Image J.

To determine if chronic inflammation in human colon might affect membrane distribution of the alpha subunit of the ATPase, paraffin sections from normal human colon, Crohn’s disease, ulcerative colitis, and colon cancer were examined. To better observe the staining, small sections of each figure are enlarged to the outside of each panel. In normal colon, the alpha subunit of the Na+-K+-ATPase was stained exclusively the basolateral membrane of the colonocytes (Figure 8). Similarly, the alpha subunit of the Na,K-ATPase was exclusively found in the basolateral region membranes of colonocytes found in a section from a patient with colon cancer (sporadic cancer). However, in several regions of active inflammation found in sections from Crohn’s disease or ulcerative colitis, the alpha subunit could be detected on the apical membrane, suggesting that the fence function of the tight junctions may be compromised in these human diseases.


Figure 8.

Na+-K+-ATPase alpha subunit is found in the apical membrane in human

inflammatory bowel diseases. Sections were obtained from human colon without disease (A), with sporadic cancer (B), with active Crohn’s disease (C) or ulcerative colitis (D), and stained for the alpha subunit of the ATPase.

Images shown are representative of at

least five regions observed on the same slide and two different sections were analyzed for each condition. Enlargements to the outside were selected from the images presented to demonstrate apical Na+-K+-ATPase subunit staining which is designated by arrows. Results are based on a double blinded experiment.

DISCUSSION Reactive oxygen species (ROS) are major mediators of the inflammatory process and are produced in large quantity in inflammatory bowel diseases by infiltrating


inflammatory cells and resident macrophages.

Of the forms of ROS, chloramines,

including NH2Cl, are physiologically relevant because of their rapid formation from immune cell-derived hypochlorous acid and amine containing molecules, including gut flora-derived ammonia. Because it is long lived and cell permeant, NH2Cl is highly cytotoxic, affecting multiple targets including membrane lipids, cellular proteins, and nucleic acids. Several studies have now shown that ROS may play a major role in compromised epithelial transport and barrier function associated with mucosal inflammation (1, 12, 20-21, 33-36, 44).

This is particularly true under the stress

conditions of acute inflammation leading to selective downregulation of barrier and transport-related protein expression (18, 48). This would suggest specific targeting of cellular targets of the apical barrier related proteins of the tight junction, such as ZO-1 and occludin, resulting in dysfunction. A number of second messenger pathways may be involved in the oxidant-induced disruption of tight barrier function. Studies have shown that xanthine/xanthine oxidase system (which generates predominantly superoxide) or acetaldehyde (which generates a number of reactive oxygen metabolites) (1) decrease monolayer TER (4, 33).


superoxide-induced TER decrease is blocked by the tyrosine kinase inhibitor genistein (35), however the effects of oxidants on TER may also involve the regulation of phosphoprotein phosphatases such as PP2B which is inactivated under these conditions (36) and acetaldehyde which nearly eliminates PTP1B activity (1). Hydrogen peroxide stimulates the redistribution of occludin and ZO-1, an event that is dependent on activation of c-src kinase (4). Additionally, tyrosine phosphorylation of occludin may also influence its interaction with ZO-2 and –3 (17). Oxidants decrease cellular levels of


glutathione, which also regulates protein phosphatase activity (34) and could have additional effects on TER via other pathways that regulate tight junctional protein distribution and function. The present results demonstrate that the physiologically relevant, long-lived, and cell permeant oxidant NH2 Cl causes alterations both in the cellular localization and levels of occludin and ZO-1. Concomitantly, NH2Cl stimulates disruption of actin filaments, including the perijunctional actin (27) using concentrations that fall within the estimated range observed at sites of tissue inflammation (12). The gate and fence tight junctional functions may not be similarly regulated via modulation of ZO-1 and occludin distribution and expression and the state of actin. We used the actin filament stabilizer phalloidin to prevent the NH2Cl-induced depolmerization of actin. While this paradigm was not effective in preventing NH2Cl-induced changes in TER, the redistribution and decreased expression of ZO-1 and occludin as well as movement of the basolateral alpha subunit of the Na+-K+-ATPase were both largely inhibited by phalloidin pretreatment suggesting a differential regulation of these events. We also used the actin depolymerizer cytochalasin D to address the roles of actin in regulating gate and fence functions. While cytochalasin D stimulates rapid and large decreases of TER of equal magnitude to those with NH2Cl, cytochalasin D stimulated slower and more modest changes in distribution and expression of ZO-1 and occludin and only after prolonged incubations was there movement of the alpha ATPase subunit to the apical membrane, an indication of altered cellular polarity.

These results suggest complex regulation of both gate and fence

function. Phalloidin was able to prevent NH2Cl-stimulated redistribution of occludin and ZO-1, but less able to prevent actin disruption by the oxidant and was not able to prevent


NH2Cl-stimulated changes in TER and mannitol flux. It appears that presence of ZO-1 and occludin in the NP-40 insoluble fraction, possibly at the tight junction, are not sufficient to maintain the gate function of the tight junctions. Cytochalasin D, which stimulates a rapid decrease in gate function but only a more modest and delayed change in fence function might suggest that the perijunctional actin plays a modest role in the fence function. These hypotheses must be interpreted with caution as it is known that the perijunctional actin associates with ZO-1 and perhaps to other proteins of the tight junctional complex. The important prolonged maintenance of ZO-1 at the tight junction may be regulated not only by association with actin, but also association with occludin and possibly other proteins of this multi-protein complex. It should be noted that differences in gate and fence functions have been previously described (43).

The actin depolymerizer mycalolide B increases paracellular

permeability (gate function) with little change in fence function. Silencing RNA of occludin had no effect on the fence function of MDCK II monolayers as assessed by BODIPY-labeled sphingomyelin in the apical membrane (50). Notable in these studies was that membrane cholesterol depletion, which decrease TER and stimulates actin cytoskeletal reorganization, was prevented in occludin-deficient cells due to a lack of occludin modulation of Rho-GTP activity. With regards to gate function in these studies, although no changes in basal TER were noted, permeability to monovalent organic anions increased (although no changes in Cl- or mannitol monolayer permeability were noted). Therefore, at present, both the formation and regulation of the fence, as well as the gate functions are incompletely understood. The roles of the integral membrane proteins occludin and the claudins, as well as the adherens junctions and a pivotal protein


of this junction, JAM-1 are also hypothesized to play a role in the regulation of the fence function (8, 19). Alterations in fence function may not necessarily been seen for all plasma membrane proteins including the distribution of the sodium/hydrogen exchanger. NHE3 was not affected by either cytochalasin D or NH2Cl treatment. We speculate that some membrane proteins such as NHE3 are tethered by additional mechanisms or factors, which are oxidant-resistant and maintain polarity. Conversely, the apical membrane protein GP135 shows lateral mobility into the basolateral membrane following rotaviral infection (28). It would therefore appear that alterations in the fence function may have a profound effect on distribution or may have no effect at all. In summary, this study provides important insights into the mechanisms of oxidantmediated decreases in intestinal epithelial barrier function. Our data indicate that ROS target perijunctional actin and both the levels and distribution of key tight junctionassociated proteins. Oxidant disruption of perijunctional actin is not by itself sufficient to cause these effects; rather a combination of events mediates the oxidants actions. Additionally, oxidant-induced loss of barrier function is associated with compromised fence function. The latter can cause depolymerization of critical membrane proteins, which is capable of severely disturbing many epithelial functions, particularly vectorial transport of nutrients and electrolytes. This prospect could contribute to development of malabsorption and diarrhea in patients with mucosal inflammation.


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