Increased cysteine-protease activity in fecal supernatants from constipated IBS-C patients leads to occludin enzymatic degradation in colonic mucosa

• Published in: Zeitschrift für Gastroenterologie
• Main Authors: Róka, R, Annaházi, A, Bezirard, V, Eutamene, H, Ferrier, L, Ducrotté, P, Inczefi, O, Rosztóczy, A, Ollé, G, Gecse, K, Molnár, T, Piche, T, Theodorou, V, Bueno, L, Wittmann, T
• Format: Conference Proceeding
• Language: English; German
• Published: 06.05.2011
• ISSN: 0044-2771; 1439-7803
• DOI: 10.1055/s-0031-1278504

Background and aims: We have previously demonstrated that (i) fecal cysteine-protease (CP) activity is elevated only in constipated IBS (IBS-C) patients among IBS subtypes and (ii) repeated mucosal exposure to this luminal factor enhances colorectal sensitivity and increases intestinal permeability in mice via the degradation of occludin. We aimed to further explore if IBS-C fecal supernatant is able to degrade the recombinant tight junction protein, occludin in vitro and if occludin levels are decreased in colonic biopsies from IBS-C patients. Methods: Fecal samples and colonic biopsies were collected from healthy subjects (n=20 and 5 respectively) and IBS-C patients (n=25 and 5 respectively). CP activity was assayed using a selective substrate and controlled by a selective CP inhibitor, E64. Recombinant occludin was incubated in presence of fecal supernatants from IBS-C patients or healthy controls or papain (as a positive control of cysteine-proteases) during 15min, and occludin degradation was assessed by western blotting. Occludin expression was determined in colonic biopsies from IBS-C patients and healthy controls. Results: CP activity in IBS-C FSN was significantly increased (p<0.05) vs. healthy controls (2420±718 and 319±91 Δfluo/mg prot). IBS-C FSN and papain degraded recombinant occludin by 72 and 95% respectively vs. healthy controls, an effect partly reversed by E64. Occludin protein levels were 2-fold lower in IBS-C colonic biopsies vs. healthy controls. Conclusion: Our results confirm that elevated cysteine-protease activity found in IBS-C fecal supernatant is able to cleave occludin by enzymatic degradation, and occludin expression is decreased in IBS-C colonic biopsies, which may explain the increased permeability shown previously by animal studies. These data suggest that the elevated fecal cysteine-protease activity may play a role in the pathogenesis of IBS-C by increasing colonic permeability, which may lead to a mucosal microinflammation and hypersensitivity. TÁMOP-4.2.2–08/01–2008–0002