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Activation of colonic proteinase-activated receptor-2 (PAR2) caused inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR2-activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 μg per mouse, in an attempt to discriminate between both PAR2-mediated effects. We further investigated the possible involvement of interferon γ (IFN-γ) and calmodulin-dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens-1 (ZO-1) localization in PAR2-induced responses. Thus, at the lower dose, SLIGRL increased colonic permeability without causing inflammation. Western blotting showed phosphorylation of mucosal myosin light chain (MLC) expression after both doses of SLIGRL. Moreover, while the MLCK inhibitor, ML-7, abolished the permeability effects of the low dose of SLIGRL, it only partially inhibited that of the high dose. In IFN-γ-deficient mice (B6 ifng−/−), the increases in permeability were similar for both doses of SLIGRL and prevented by ML-7. In addition, MLCK immunoprecipitation revealed an increase of calmodulin binding to MLCK in the mucosa of mice treated with either dose of SLIGRL. Finally, we have shown that direct activation of PAR2 on enterocytes is responsible for increased permeability and ZO-1 disruption. Moreover, SLIGRL at a dose that does not produce inflammation increases permeability via calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN-γ secretion, while the increased permeability in response to the high dose of PAR2 agonist involves IFN-γ secretion.
A major function of gastrointestinal epithelial cells is to form a physical barrier between the gastrointestinal lumen and subepithelial tissues. The apico-laterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells. Lateral contacts, which may be visualized by electron microscopy and freeze-fracture analysis, act as a structural barrier against paracellular permeation (Jinguji & Ishikawa, 1992). Epithelial TJs are composed of at least three families of transmembrane proteins (occludins, claudins and adhesion proteins) and a cytoplasmic ‘plaque’ consisting of many different proteins that form large complexes. The transmembrane proteins mediate cell adhesion and are thought to constitute the intramembrane and paracellular diffusion barriers. The cytoplasmic plaque of TJs is formed by different types of proteins that include adaptors, such as the zonula occludens (ZO) proteins and other proteins that contain PDZ domains, as well as regulatory and signalling components. Adaptor proteins are thought to be connected to transmembrane proteins and to recruit other cytosolic components to TJs, such as protein kinases, GTPases and transcription factors (Mitic & Anderson, 1998). Several components of the cytoplasmic plaque, in addition to the transmembrane protein occludin, also function as cytoskeletal linkers that interact with the cytoskeleton (Mitic & Anderson, 1998). There is a high density of cytoskeletal actin and myosin filaments, which encircle the intestinal epithelial cells near the apical cellular borders at the level of TJs. The disruption of perijunctional actin–myosin filaments allows an increase in epithelial penetration (Yamaguchi et al. 1991). The activity of myosin is regulated by the opposing actions of myosin light-chain phosphatase and myosin light-chain kinase. Myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) is one of the regulators of TJ permeability (Turner et al. 1997). In mice, stress-induced opening of colonic epithelial TJ requires CD4+/CD8+ T cells and IFN-γ, and involves MLCK activation (Ferrier et al. 2003). In the colon, a leaky TJ barrier allows penetration of toxic luminal substances, which promote colonic mucosal injury and inflammation (Gassler et al. 2001).
Proteinase-activated receptors (PARs) belong to a family of seven transmembrane domain G-protein-coupled receptors that are activated by cleavage of their N-terminal domain by a proteolytic enzyme (Nystedt et al. 1995). The unmasked new N-terminal sequence acts as a tethered ligand that binds and activates the receptor itself. PAR2 is activated by trypsin, mast cell tryptase and trypsin-like proteins. Synthetic peptides, so-called PAR-activating peptides (PAR-APs) such as SLIGRL for PAR2, corresponding to the amino-acid sequence of the tethered ligand, are also able to selectively activate PARs (Corvera et al. 1997). PAR2 is found expressed throughout the gastrointestinal tract on several cell types, including enterocytes, mast cells, smooth muscle cells, myenteric neurones and endothelial cells (Kong et al. 1997). Moreover, the involvement of PAR2 in different pathophysiological processes has been reported. In fact, in vivo intracolonic activation of PAR2 led to colonic inflammation in mice and increased paracellular permeability, with bacterial translocation into peritoneal organs (Cenac et al. 2002). In this model, colonic infusion of trypsin, tryptase or SLIGRL induced a Th1-like inflammation, in which TNF-α, interleukin (IL)-1β and IFN-γ mRNA levels are elevated, while IL-4 and IL-10 mRNA expression remain stable (Cenac et al. 2002). In vitro, it has been shown that IFN-γ disrupts the epithelial barrier function of T84 cells by decreasing the levels of ZO-1, perturbing the actin cytoskeleton in the tight junction area, and displacing ZO-2 and occludin (Youakim & Ahdieh, 1999). In addition, TNF-α has been reported to promote the disturbing effects of IFN-γ on the epithelial barrier (Mahraoui et al. 1997), while IL-10 blocks these effects (Oshima et al. 2001). Although the expression of PAR2 has been reported on both apical and basolateral surfaces of enterocytes, evidence is lacking so far that direct activation of PAR2 may lead to a dysfunction of the epithelial barrier.
Consequently, the aim of this work was to determine the mechanisms underlying the effects of different concentrations of a PAR2 agonist, the peptide SLIGRL, on colonic paracellular permeability. Specifically, we investigated (i) the involvement of IFN-γ, (ii) the involvement and activation pathway of MLCK, and (iii) the direct effects of PAR2 agonist on enterocyte barrier functions.
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Our study reveals for the first time that (i) a low dose of SLIGRL increases paracellular permeability via MLC phosphorylation by MLCK without causing inflammation, and (ii) that the increase in paracellular permeability mediated by the inflammatory dose of SLIGRL involves an INF-γ-dependent mechanism. Activation of PAR2 by low doses of SLIGRL provoked the activation of calmodulin, which interacts with and stimulates MLCK most likely in epithelial cells, without concomitant influence of inflammatory factors. In fact, for all strains of mice studied here, low doses of PAR2-AP had no effect on inflammatory parameters. By electron microscopy, we have shown that a low dose of SLIGRL opened epithelial cell tight-junctions with a concurrent activation of MLCK, which in turn, led to an increase in p-MLC proteins observed by Western-blot analysis. Moreover, PAR2 agonist relocalized ZO-1 and increased paracellular permeability in epithelial monolayers, further demonstrating a direct effect of PAR2 activation on enterocytes. In C57BL/6J and Swiss 3T3 mice, permeability was dose-dependently increased after SLIGRL. While ML-7 abolished the effect of the low dose of SLIGRL, it only decreased that of the high dose. In these mice, high doses of SLIGRL provoked an inflammatory reaction characterized by increased MPO activity, high damage scores, increased IFN-γ and TNF-α mRNA, and decreased IL-10 mRNA. In IFN-γ-deficient mice, SLIGRL had no effect on inflammatory parameters, but increased intestinal permeability to the same extent for both doses of SLIGRL. This increase in paracellular permeability was linked to MLCK activation. The inflammatory reaction is IFN-γ dependent and for the high dose of SLIGRL the paracellular permeability is IFN-γ and calmodulin–MLCK dependent. In contrast, for the low dose of SLIGRL, the increase in paracellular permeability depends on calmodulin–MLCK activation, but not on IFN-γ.
In previous studies, we have shown that the inflammatory reaction mediated by high doses of SLIGRL was inhibited by chronic pretreatment with either capsaicin, or by NK1 or CGRP receptor antagonist suggesting a neurogenic component of this inflammation (Cenac et al. 2003; Nguyen et al. 2003). While the inflammatory response was inhibited by such treatments, a residual increase of paracellular permeability was still observed and was of the same magnitude as the increase observed for a low dose of SLIGRL (Cenac et al. 2003). The calmodulin–MLCK complex, as well as inflammatory mediators such as IFN-γ are involved in SLIGRL-induced paracellular permeability increase during inflammation. Indeed, in B6 ifng−/− mice, the increase in permeability was similar for both doses of SLIGRL and prevented by ML-7. The high dose of SLIGRL did not increase MPO activity, damage score, or TNF-α and IL-10 mRNA in these IFN-γ-deficient mice.
The increased permeability response to PAR2 activation may not be linked to selective activation of PAR2 located at the apical site of epithelial cell membrane as in vitro, only when PAR2 agonist was added to the basolateral side of SCBN monolayers, permeability to FITC–dextran was increased. However, it is not known whether PAR2 presents the same distribution on apical and basal sides of epithelial cell membranes in vitro and in vivo. We hypothesize that the non-inflammatory permeability response to PAR2 activation is linked to the activation of a subset of receptors, with distinct intracellular pathways. Indeed, in human airway smooth muscle cells, PAR2 activation stimulates phospholipase C (PLC) via its coupled Gq protein (Berger et al. 2001). The activation of the PLC-dependent signalling pathway has been implicated in the assembly, regulation and barrier properties of the tight junction (Balda et al. 1991). In fact activation of PLC releases IP3 into the cytosol, which increases intracellular Ca2+ concentration, which activates MLCK through the activation of calmodulin-dependent kinases (Berridge et al. 1983; Streb et al. 1983). MLCK phosphorylates MLC and thereby induces contraction of the perijunctional actin–myosin ring, which has been reported to provoke the opening of tight junctions (Yamaguchi et al. 1991). The increase in paracellular permeability caused by ethanol (Ma et al. 1999) and medium chain fatty acids, such as capric and lauric acid (Lindmark et al. 1998), as well as that caused by microbes (Scott et al. 2002) are attributed to this mechanism. Ethanol caused disassembly and displacement of perijunctional actin–myosin ring and stimulates MLCK (Ma et al. 1999). The inhibitor of MLCK (ML-7) attenuated both the ethanol-mediated increase in paracellular permeability and MLCK activity (Ma et al. 1999). Similarly, chelation of cytosolic Ca2+ and inhibition of MLCK attenuated the ability of capric and lauric acid to increase paracellular permeability (Lindmark et al. 1998). In our study we show that the activation of PAR2 increases the interaction of calmodulin and MLCK probably by an increase of intracellular Ca2+ concentration. This hypothesis is further supported by the fact that in many cell types, PAR2 activation causes intracellular calcium mobilization. This complex favours MLC phosphorylation and perijunctional actin–myosin ring contraction leading to alteration of paracellular permeability.
At a higher dose, PAR2-AP may be absorbed, and subsequently activates receptors located at the baso-lateral site of epithelial cells or other cell types such as immunocytes or nerves, inducing a neurogenic inflammation (Cenac et al. 2003; Nguyen et al. 2003). In agreement with this hypothesis, capsaicin inhibits the inflammatory reaction and diminishes the increase in permeability (Cenac et al. 2003). Results obtained in IFN-γ-deficient mice confirmed the crucial role of IFN-γ since no inflammation was seen even with a high dose of PAR2-AP in these animals. Disruption of the epithelial barrier in the gut is a hallmark of inflammatory bowel disease (IBD) and intestinal infections (Gitter et al. 2001). Although it remains unclear whether barrier breakdown is an initiating event or a consequence of inflammation, it is obvious that barrier loss contributes to propagation and exacerbation of inflammation (Gassler et al. 2001). Barrier breakdown can be elicited by a number of agents, including bacteria, immunocytes, and proinflammatory cytokines. Previous studies have demonstrated that T84 cells exposed to IFN-γ show decreased barrier function integrity (Youakim & Ahdieh, 1999). IFN-γ is greatly elevated in human intestinal disease and undoubtedly contributes to the inflammatory cascade, which includes barrier disruption. In fact, IFN-γ acts on at least two elements critical to barrier function. The first is to cause a decrease in the expression of ZO-1, a key component of the tight junction (Youakim & Ahdieh, 1999). The second is to alter the organization of the actin cytoskeleton in the apical region of the cells (Youakim & Ahdieh, 1999). TNF-α can enhance these effects, probably due to IFN-γ-dependent increase in TNF receptor gene expression suggesting that, in IBD, TNF-α may have synergistic effects on IFN-γ-mediated alterations of epithelial cell function (Rodriguez et al. 1995). In a recent study carried out on 23 patients with active Crohn's disease, it was demonstrated that treatment with TNF antibodies largely restores the gut barrier (Suenaert et al. 2002). Recent clinical trials provided evidence for a therapeutic effect of IL-10 in patients with Crohn's disease by reducing intestinal inflammation (Schmit et al. 2002). One potential explanation for the observed inhibition of IFN-γ effects on barrier function by IL-10 could be that IL-10 down-regulates receptors for IFN-γ (Oshima et al. 2001). A beneficial effect of IL-10 is its ability to block the reduced expression of occludin induced by IFN-γ (Oshima et al. 2001).
PAR2 activation at an ‘inflammatory dose’ of SLIGRL was associated with an increase in TNF-α, IFN-γ and IL-1β expression and a decrease in IL-10 expression. Thus, we can hypothesize that the Th1 profile mediated by PAR2 activation during the inflammatory reaction increased paracellular permeability by a combined action of IFN-γ and TNF-α on the expression of ZO-1 and on the organization of the actin cytoskeleton in the apical region. This hypothesis is supported by the reduction of paracellular permeability increase in mice deficient for IFN-γ.
In summary, in non-inflammatory conditions, PAR2 activation increases paracellular permeability through calmodulin activation, which can bind to and activate MLCK, and then provokes tight junction opening by perijunctional ring myosin phosphorylation. In inflammatory conditions increased paracellular permeability is mediated by the pathway described above and by an IFNγ-dependent pathway.