PAR2 activation alters colonic paracellular permeability in mice via IFN-gamma-dependent and -independent pathways

Abstract

Activation of colonic proteinase-activated receptor-2 (PAR(2)) 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 PAR(2)-activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 microg per mouse, in an attempt to discriminate between both PAR(2)-mediated effects. We further investigated the possible involvement of interferon gamma (IFN-gamma) and calmodulin-dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens-1 (ZO-1) localization in PAR(2)-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-gamma-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 PAR(2) 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-gamma secretion, while the increased permeability in response to the high dose of PAR(2) agonist involves IFN-gamma secretion.

Figure 1. MLCK, MLC and p-MLC and electron microscopy of tight junctions

A, detection of MLCK, MLC and p-MLC proteins by Western blot. Mice (Swiss 3T3) received intracolonocally SLIGRL (5 and 100 μg per mouse), LRGILS (100 μg per mouse) or their vehicle (saline). Mucosa lysates were incubated with anti-mouse MLCK, MLC or p-MLC antibodies. MLCK, MLC and p-MLC were detected as bands with a molecular mass of about 130, 20 and 20 kDa, respectively (n = 5). B, electron microscopy of tight junction observed in mice Swiss 3T3 colon treated with SLIGRL (5 μg per mouse), LRGILS (100 μg per mouse) or saline (n = 3).

Figure 2. Prevention by MLCK inhibitor of SLIGRL-induced intracellular permeability increase and MLC phosphorylation

A, prevention by the MLCK inhibitor, ML-7, of SLIGRL-induced colonic paracellular permeability increase. B6 ifng^−/− mice (IFN-γ deficient) and their control strain (C57BL/6J) were pretreated by ML-7 (2 mg kg⁻¹) and received intracolonic administration of saline, LRGILS (100 μg per mouse) or SLIGRL (5 and 100 μg per mouse). Intestinal permeability was assessed by measuring the gut lumen-to-body passage of labelled ⁵¹Cr-EDTA. Values are means ±s.e.m.; n = 8; *P < 0.05 compared to ‘saline’ values, #P < 0.05 compared to the values of the group treated by 5 μg per mouse of SLIGRL. B, detection of MLCK, MLC and p-MLC proteins by Western blot. IFN-γ-deficient and wild-type mice were treated by SLIGRL (5 and 100 μg per mouse) or its vehicle (saline) and pretreated by a MLCK inhibitor, ML-7 (2 mg kg⁻¹, i.p.), or its vehicle (2% ethanol). Mucosa lysates were incubated with anti-mouse MLCK, MLC or p-MLC antibodies. MLCK, MLC and p-MLC were detected as bands with a molecular mass of about 130, 20 and 20 kDa, respectively (n = 5).

Figure 3. Interaction between MLCK and calmodulin

A, inhibition by the calmodulin inhibitor, chlorpromazine, of SLIGRL-induced colonic paracellular permeability increase. Swiss 3T3 mice were pretreated by chlorpromazine (25 mg kg⁻¹, i.p.) (grey bars) or its vehicle (saline) (black bars) and received intracolonically saline, LRGILS (100 μg per mouse) or SLIGRL (5 and 100 μg per mouse). Intestinal permeability was assessed by measuring the gut lumen-to-body passage of labelled ⁵¹Cr-EDTA. Values are means ±s.e.m.; n = 8; *P < 0.05 compared to ‘saline’ values. B, calmodulin–MLCK interactions. Four hours after treatment of Swiss 3T3 mice by saline, LRGILS (100 μg per mouse) or SLIGRL (5 and 100 μg per mouse), colons were removed, and colonic mucosa proteins were immunoprecipitated with an anti-MLCK antibody followed by calmodulin and MLCK immunoblotting assays. MLCK and calmodulin were detected as bands with a molecular mass of about 130 kDa and 16 kDa, respectively (n = 5).

Figure 4. Paracellular permeability of SCBN monolayers, measured as transepithelial flux of FITC-dextran 3000

Values were obtained from control monolayers with vehicle and monolayers incubated with SLIGRL (30 μm) or LRGILS (30 μm) for 2 h (apical, A, and basal, B). Values are means ±s.e.m. of the percentage apical fluorescence that crossed 1 cm² of the epithelial barrier into the basolateral chamber in 1 h. *P < 0.05 compared to saline values; n = 12 per group.

Figure 5. Representative confocal laser scanning micrographs of SCBN monolayers stained for tight junctional ZO-1

A, preparations included unmanipulated control monolayers: typical distribution of ZO-1 in control monolayers can be seen at the periphery of the enterocytes; B, monolayers exposed to SLIGRL (30 μm) for 2 h: exposure to SLIGRL causes a perinuclear relocation of ZO-1; C, monolayers exposed to LRGILS (30 μm) for 2 h: LRGILS had no effect.