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. 2009 Jul;175(1):207-14.
doi: 10.2353/ajpath.2009.080986. Epub 2009 Jun 15.

Luminal cathepsin g and protease-activated receptor 4: a duet involved in alterations of the colonic epithelial barrier in ulcerative colitis

Marta Dabek  1 , Laurent Ferrier, Richard Roka, Krisztina Gecse, Anita Annahazi, Jacques Moreau, Jean Escourrou, Christel Cartier, Gilles Chaumaz, Mathilde Leveque, Afifa Ait-Belgnaoui, Tibor Wittmann, Vassilia Theodorou, Lionel Bueno
Affiliations

Luminal cathepsin g and protease-activated receptor 4: a duet involved in alterations of the colonic epithelial barrier in ulcerative colitis

Marta Dabek et al. Am J Pathol. 2009 Jul.

Abstract

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR(4), in these processes. Expression levels of both PAR(4) and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR(4) was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR(4)-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR(4) was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR(4) play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

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Figures

Figure 1
Figure 1
A: Expression of Cat-G in biopsy samples from healthy (H) (n = 6) and UC (U) (n = 7) subjects. B: Western blotting of Cat-G in fecal supernatants from UC patients (n = 8) and healthy subjects (n = 7). C: Densitometry analysis of Cat-G expression in biopsy samples normalized to GAPDH. D: The integrated density values of Cat-G in fecal supernatants. Data are expressed as means ± SEM, *P < 0.05, ***P < 0.001 compared with healthy subjects.
Figure 2
Figure 2
Immunohistochemical staining of longitudinal (A, B) and transverse serial section (C, D) for PAR4 expression (arrowheads) in human colonic biopsies from healthy subjects (n = 7) (A, C) and UC patients (n = 6) (B, D). RT-PCR was performed on similar samples (E), and expression of PAR4 was normalized to GAPDH and analyzed by densitometry by using ImageJ software (F). Data are expressed as means ± SEM (n = 6/7), *P < 0.05 compared with healthy subjects.
Figure 3
Figure 3
A: Expression of PAR4 in colonic mucosa from wild-type C57Bl.6/J mice (n = 6). GAPDH served as control. B: Colonic paracellular permeability (CPP) to FITC-Dextran in Ussing chambers 60 minutes after the administration of fecal supernatants of UC patients (n = 7), CD patients (n = 6), healthy controls (n = 6), and saline (n = 10) on colonic strips of wild-type mice, and CPP after prior incubation of fecal supernatant of UC patients (n = 15), CD patients (n = 7), healthy controls (n = 12), and saline (n = 12) with pepducin P4pal-10 targeting PAR4 (N-palmitodyl-SGRRYGHALR-NH2, 1 μmol/L). +P < 0.05 compared with fecal supernatants of UC and CD patients, *P < 0.05 compared with saline and **P < 0.01 compared with saline. C: CPP after prior incubation of fecal supernatant of UC patients (n = 6) with the PAR1 antagonist, FLLRN (10 μmol/L) and the PAR2 antagonist, FSLLRY-amide (10 μmol/L). No significant differences were observed. D: CPP 60 minutes after the administration of PAR4 agonist (AY-NH2, 50 μmol/L) (n = 10) and saline (n = 7) controls on colonic strips of wild-type mice. Data are expressed as means ± SEM, ***P < 0.001.
Figure 4
Figure 4
A: Cat-G activity assessed with the substrate N-Suc-Ala-Ala-Pro-Phe-pNA, in fecal supernatant from healthy subjects (n = 6) and UC patients (n = 9), pretreated or not with a specific Cat-G inhibitor (SCGI) (1 μmol/L). Data are expressed as means ± SEM, ***P < 0.001 compared with fecal supernatants from healthy subjects, and ++P < 0.01 compared with fecal supernatants from UC patients. B: Colonic paracellular permeability to FITC-Dextran after prior incubation of fecal supernatants of UC patients with SCGI (0.2 μmol/L). Data are expressed as means ± SEM (n = 10), *P < 0.05, **P < 0.01 compared with fecal supernatants from UC patients. C: Fecal MPO activity in mice colon 1 day after intracolonic infusion of fecal supernatants of UC patients, healthy controls, saline and UC supernatants previously incubated with SCGI. Data are expressed as means ± SEM (n = 7), *P < 0.05, ***P < 0.001 compared with UC supernatants infusion.
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