Summary

The epithelial lining of luminal organs such as the gastrointestinal and respiratory tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Paracellular permeability across epithelial and endothelial cells is in large part regulated by an apical intercellular junction also referred to as the tight junction (TJ). The tight junction and its subjacent adherens junction (AJ) constitute the apical junctional complex (AJC). The AJC is composed of a multiprotein complex, which affiliates with the underlying apical perijunctional F-actin ring. Such AJC association with the perijunctional F-actin ring is vital for maintaining its structure and function in health. Stimuli such as nutrients, internal signaling molecules and cytokines influence the apical F-actin organization and also modulate the AJC structure and paracellular permeability. Here we review some of the key stimuli that influence F-actin organization, AJC structure and paracellular permeability.

Introduction

The tight junction (TJ) is the apical most intercellular junction in epithelial and endothelial cells and in association with the subjacent adherens junction (AJ) it constitutes the apical junctional complex (AJC). TJs form regulated, selectively permeable barriers between two distinct compartments.^1,2 Thus, for example in the intestinal and respiratory tract, TJs interface luminal contents and underlying tissue compartments. TJs do not just represent static structural elements but they are dynamically regulated to control paracellular solute and ion transport in diverse physiologic states. In part such regulation in health and dysregulation in disease occurs secondary to signaling events influencing the underlying perijunctional actin cytoskeleton.^3,4

Columnar epithelial cells have a prominent perijunctional actin-myosin II ring that encircles the apical pole of polarized cells. This perijunctional filamentous actin ring is readily visualized by fluorescence labeling of filamentous actin and by electron microscopy. In fact the major interface of this ring with the lateral membrane occurs just below the TJ in the AJ. However, actin filaments project from this ring and interface with specific sites of membrane kisses in TJs that represent regions where membranes from apposing cells come into close apposition (fig. 1).⁵ Thus, it is logical to envision regulation of TJ structure and paracellular solute transport by factors that influence lateral tension within the perijunctional actin-myosin II ring.^6-8

Figure 1

Protein composition and regulation of the Apical Junctional Complex (AJC). Transmembrane proteins in the apical junctional complex affiliate with an underlying perijunctional actin myosin ring. Signaling molecules and extracellular stimuli such as cytokines, (more...)

Our knowledge of TJ protein composition and regulation of paracellular transport is rapidly expanding. It is clearly evident that transmembrane proteins in TJs such as occludin, claudin(s) and junction adhesion molecule (JAM)1 affiliate with cytoplasmic plaque proteins that in turn have been implicated in the association of the TJ protein complex with the actin cytoskeleton.^9-12 Prototypes of such actin associating proteins are the zonula occludens proteins (ZO-1, ZO-2 and ZO3). Cingulin also interacts with the zonula occludens proteins and enterocyte myosin heavy chain.¹³ The overall organization of TJ associated proteins is illustrated in Figure 1. Structure and functional properties of these proteins are detailed in other chapters of this issue.

Current knowledge of TJs is consistent with a view that they are specialized membrane microdomains^14-16 that might function as molecular platforms involved in actin organization (Rho-GTPases, EFA6), cell signaling (c-src and c-yes), membrane trafficking (VAP-33, Rab3b, Rab13, Rab 8, Sec6, and Sec8), and cell polarity (Par3 and Par6).^17-20 Thus TJs represent very dynamic platforms that regulate paracellular movement of ions and solutes in many physiologic and pathologic states. Diverse stimuli that influence TJ function and its subjacent actin cytoskeleton are discussed below.

Modulation of Myosin Light Chain-Phosphorylation in the Perijunctional F-Actin Ring Influences TJ Function

Circumferential contraction and therefore tension in the apical perijunctional actin-myosin ring regulates solute transport across the paracellular space.^21-25 Such modifications in the perijunctional actin-myosin ring are achieved by the phosphorylation of the myosin light chain (MLC) of myosin II by MLC kinase (MLCK), which in turn acts on bipolar F-actin fibers in the perijunctional F-actin ring. Increased epithelial permeability in MLCK-dependent fashion has been demonstrated in model intestinal epithelial cell lines exposed to enteropathogenic Escherichia coli, transmigrating polymorphonuclear leukocytes or cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α).^26-28 In addition, physiologic agonists have also been demonstrated to influence paracellular transport by modifications in the perijunctional actin-myosin ring. A classic scenario involves the uptake of glucose in the intestinal tract.²⁹ Early ultrastructural examination of the intestinal mucosa has demonstrated that intrajunctional dilatations and condensation of the perijunctional cytoskeleton occur with Na⁺-glucose cotransport induced increase in permeability.^22,30,31 Such modifications in the actin cytoskeleton support the concept of cytoskeletal regulation of paracellular permeability. In fact, subsequent studies linked the activation of enterocyte Na⁺-glucose cotransporter with phosphorylation of MLC.³² Na⁺-glucose cotransport induces cytoplasmic alkalinization that is dependent on the activation of the brush border Na⁺/H+ exchanger isoform NHE3.³³ Inhibition of the NHE3 exchanger reduces MLC phosphorylation that is associated with an increase in transepithelial resistance (TER) to passive ion flow.³³ It has therefore been proposed that NHE3 activation may be a critical component of the signaling pathway for Na⁺-glucose cotransport-dependent TJ regulation. Further evidence supporting the role of MLC phosphorylation in TJ regulation comes from studies using epithelial cells transfected with truncated MLCK gene construct lacking the inhibitory domain required for kinase regulation.³⁴ Expression of this construct in model epithelial cell lines induced an increase in myosin regulatory light chain phosphorylation and an increase in paracellular permeability.^27,34 Upstream regulation of the MLCK by protein kinase C has been proposed as a mechanism regulating the TJ permeability.35

Other physiological agonists influencing the cytoskeleton and junctional transport through modulation of the actin cytoskeleton include responses to histamine³⁶ and lysophosphatidic acid³⁷ that induce phosphorylation of MLCs. Ethanol and low concentration of extracellular calcium increase activity of MLCK and disrupt the TJ protein complex by influencing ZO-1 and occludin organization in the AJC.^38,39 Agonists that influence TJ permeability in rat hepatocytes include angiotensin II, vasopressin and epinephrine.⁴⁰

Modulation of Barrier Function by Rho GTPases

The Rho family of small GTPases, comprising Rho, Rac and Cdc42, are believed to play an important role in regulating and maintaining the perijunctional actin ring, TJ structure/function, and assembly of polarized epithelial cells.^41-46 Rho function is modulated by a set of regulatory proteins and is activated through GDP-GTP exchange, which is promoted by guanine nucleotide exchange factors (GEF) and is inactivated through GTPase-activating proteins.^47,48 Rho guanine nucleotide dissociation inhibitors mediate stabilization of inactive GDP-bound form of Rho.⁴⁹ Conformational changes then allow the GTPases to interact with multiple effector molecules involved in actin cytoskeletal control^50,51 Rho activity cycles are rapidly reversible, and are terminated upon hydrolysis of GTP by GTPase-activating proteins.

Several insights have been gained, based on the use of diverse pharmacological and molecular tools that interfere with function of the Rho family of GTPases. We have previously shown that inactivation of Rho GTPases (with Clostridium botulinum C3 transferase and Clostridium difficile toxins A and B) is associated with a compromised barrier function in T84 intestinal epithelial cells manifested both as functional decrease in TER, increase in paracellular flux of labeled dextran (3kDa) and a structural redistribution of ZO-1 and occludin away from the lateral plasma membrane. This effect was associated with reorganization of perijunctional F-actin.⁴⁵ Moreover, we demonstrated, that disassembly of TJs induced by Clostridium difficile toxins A and B reduced the hyperphosphorylated occludin species and ZO-1 in “raft-like” membrane microdomains.¹⁴ The downstream effector of Rho referred to as Rho kinase (ROCK) has also been documented to regulate TJ function.⁵² However, ROCK inhibition induces profound reorganization of the apical F-actin cytoskeleton without influencing TJ protein distribution in the lateral membrane. These findings imply that ROCK mediated effects on TJ function are primarily due to its influence on the apical actin cytoskeleton in epithelial cells. These observations were further supported by transfection studies, in which a dominant negative mutant of ROCK induced loss of the apical F-actin—rich brush border and a reduction in the apical perijunctional F-actin ring without influencing occludin localization. Studies using transfected epithelial cell lines expressing dominant negative mutants of Rho GTPases demonstrated increase in paracellular permeability without influencing the TJ protein organization.⁵³ In addition to the above downstream effector, an upstream GEF of the Dbl family of proto-oncogenes that activates Rho has been shown to associate with TJs.⁵⁴ A link between the TJ cytoplasmic plaque protein ZO-3 and RhoA related signaling has been proposed.¹⁷ These studies reported that transfection of the amino terminal half of ZO-3 (NZO-3) in MDCK cells resulted in decreased RhoA GTPase activity and a change in cellular F-actin organization. The authors proposed a model whereby altered interactions between ZO-3 and an AJ protein, p120 catenin in NZO-3 expressing cells influences RhoA GTPase activity.

Recent studies reported, that not only inactivation, but also activation of the Rho family of proteins enhances paracellular permeability.^43,53,55 Using an elegant inducible transfection system in MDCK cells, convincing evidence for an involvement of RhoA, Rac1 and Cdc42 in regulation of epithelial barrier function has been obtained.^53,55,56 In this system, induction of dominant-active RhoA, Rac1 and Cdc42 activity was correlated with inability of epithelial cells to develop high TER. Moreover, increased paracellular permeability to molecules of different sizes was accompanied by redistribution of occludin and ZO-1 from the lateral membrane as well as modifications in junctional associated F-actin cytoskeleton. Using the same system, we found that activation as well as inactivation of RhoA, Rac1 or Cdc42 induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. Constitutive activation of Rho A and Cdc42 induced redistribution of occludin, ZO-1, claudin-1, claudin-2 and JAM-1 from the lateral membrane. Constitutively active Rac1 on the other hand primarily influenced claudin-1 and -2 organization in TJs. These structural alterations were accompanied by changes in the biochemical properties of the TJ proteins.⁹⁷ Interestingly, an increased activation of RhoA has been described in biopsies of patients with Crohn's disease indicating that RhoA may be involved in the cascade that leads to impaired barrier function in these patients.⁵⁷ Reported effects of the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor (CNF)-1 on epithelial TER have to date been diverse. While one report suggests a lack of CNF-1 effect on TER,⁵⁸ two other reports using intestinal epithelial cell lines T84 and Caco2 document CNF-1 induced an increase in paracellular permeability.^43,59 In the latter study, increased paracellular permeability was associated with significant redistribution of the TJ proteins occludin, ZO-1, claudin-1 and JAM-1 following basolateral exposure of epithelial cells to CNF- 1. In parallel, CNF-1 incubation resulted in decreased apical F-actin that was accompanied by formation of prominent basal F-actin cables.⁴³ Thus, increased activation of Rho appears to disrupt the continuity between adjacent F-actin pools in microvilli, perijunctional ring and the terminal web that in turn could destabilize the “scaffold” of the TJ protein complex.⁴³

Interestingly, CNF-1 treatment induced internalization of TJ proteins into endosomal/ caveolar-like membranous structures, evidenced by colocalization of TJ proteins with caveolin-1 by immunogold electron-microscopy.⁴³ By immunolabeling and confocal microscopy TJ proteins were observed to colocalize with internalized early and recycling endosomal markers (EEA-1, Rab-11). This provides novel evidence that increased activation of Rho-GTPases induces internalization of TJ proteins into endosomal structures. Interestingly, dominant-active Rac1 and Cdc42 have been shown to affect endocytic trafficking in epithelial cells.^55,60,61 The colocalization of TJ proteins with markers of recycling endosomes also suggests that recycling of TJ proteins back to the lateral membrane could occur, thereby providing a route for the rapid reestablishment of barrier function during the recovery phase following injury and internalization of TJ proteins. Given that inactivation as well as activation of Rho-GTPases adversely affects epithelial barrier function, it is likely that a delicate balance of Rho activity/quiescence is required for the maintenance of the optimal epithelial/endothelial barrier function.

Modulation of Barrier Function by Cytokines

Many cytokines have been shown to influence epithelial TJ function and the actin cytoskeleton both in vivo and in vitro. The cytokines IL-1, IL-4, IL-10, IL-13, TNF-α, and IFN-γ have all been shown to regulate TJs of both epithelia and endothelia.^62-64 In addition, IL-1β influences TJ permeability through an effect on the claudin family of transmembrane proteins thought to be important in maintaining junctional integrity in astrocytes.⁶⁵ A complete review of all the cytokines shown to modulate epithelial barrier function is beyond the scope of this article. Our review therefore has focused on the influence of few select cytokines on TJ structure/function and its adjoining actin cytoskeleton.

Interferon-gamma

IFN-γ is a 20- to 25-kDa glycoprotein released by activated T cells and natural killer cells in inflammatory states. In vitro models have been extensively used to examine the influence of this pro-inflammatory cytokine on intercellular junctions of epithelial cells. The initial studies addressing the influence of this cytokine on TJs utilized model epithelial cell line, T84.^63,66,67 These studies demonstrated that IFN-γ induced a time-dependent increase in paracellular permeability that was accompanied by disorganization of apical F-actin and loss of ZO-1 from TJs.^63,68 These morphological effects were associated with a change in the differential detergent solubility profiles of ZO-1 and ZO-2. The investigators did not observe IFN-γ induced change of phosphorylation status of these proteins. This was unexpected as phosphorylation status of TJ proteins is considered to modulate assembly of the TJ protein complex.⁶⁹ Using the model T84 intestinal epithelial cell line, we have recently reported a IFN-γ-induced disruption of epithelial gate and fence function that was associated with differential internalization of TJ transmembrane proteins occludin, JAM-1, Claudin-1 and —4.⁷⁰ We have also observed a concomitant reorganization of apical F-actin (our unpublished results). In contrast ZO-1 maintained its localization in the TJs. It is intriguing to speculate why ZO-1 localization and expression levels in our study were only slightly affected by IFN-γ. ZO-1 is a key TJ cytoplasmic plaque protein that provides a scaffold upon which other proteins can be assembled.^71,72 We hypothesized that ZO-1 maintains its localization to provide this scaffold for efficient reassembly of TJ proteins upon cytokine withdrawal, which would be required for rapid and critical reestablishment of epithelial barrier function. Recent results from our laboratory support a IFN-γ induced internalization of TJ proteins into endosomal structures and this event is in part mediated by restructuring of the apical actin cytoskeleton (unpublished observations) (fig. 2). A similar mechanism of TJ protein endocytosis has been observed following depletion of extracellular calcium and disassembly of TJs.⁷³ Thus, the actin cytoskeleton appears to be essential in not only maintaining a functioning TJ but is also required for the regulated disassembly and reassembly of the TJ.

Figure 2

Colocalization of internalized occludin with the early endosomal marker EEA-1. T84 cell monolayers incubated for 48 h with IFN-γ were double-stained for occludin (green) and the early endosomal marker, EEA-1 (red) and analyzed by confocal microscopy. (more...)

An inflammatory response is regulated by a complex array of inhibitory and stimulatory cytokines, and thus it is likely that effects produced by IFN-γ are modulated by other cytokines. Several studies using different epithelial cell lines have shown, that TNF-α, another pro-inflammatory cytokine, can act synergistically with IFN-γ to increase paracellular permeability, ^27,70,74,75 most likely due to TNF-α-induced up-regulation of the IFN-γ receptor. Coyne et al⁷⁴ demonstrated in human epithelial airway cells, that combined treatment of TNF-α and IFN-γ induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. These studies emphasized the importance of the link between the actin cytoskeleton and TJs in regulation of barrier function both in the baseline state and following exposure to pro-inflammatory cytokines such as IFN-γ and TNF-α. In contrast to TNF-α, TGFβ or IL-10 have a negative influence on the IFN-γ induced changes in paracellular permeability.^76,77

The above-described effects of IFN-γTNF-α on TJs, although complex, might have pathophysiological relevance because an increase in paracellular permeability across intestinal epithelial cells has been observed in patients with inflammatory bowel diseases (IBD).⁷⁸ Since enhanced paracellular permeability across intestinal epithelium also occurs in first-degree relatives of patients with Crohn's disease, altered TJ permeability may be a contributing factor in this process,^79,80 whereas disturbed barrier function in patients with ulcerative colitis is more likely secondary to the array of inflammatory signals that characterize this state.^79,80 In this regard it is important, that redistribution of AJC proteins has been observed in tissues from patients with active IBD.^81,82

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