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HELICOBACTER PYLORI INDUCES TIGHT JUNCTIONAL CLAUDIN-4 DISRUPTION IN HUMAN GASTRIC EPITHELIAL CELLS IN A RHO KINASE-DEPENDENT MANNER
TK Lapointe1, PM O’Connor1, D Menard2, AG Buret1
1Biological Sciences & Inflammation Research Network, University of Calgary, Calgary, Alberta; 2Anatomie et Biologie Cellulaire, Université de Sherbrooke, Sherbrooke, Quebec
Epithelial tight junctional complexes play a central role in maintaining gastrointestinal barrier function. The small Rho GTPases/Rho kinase (ROCK) signaling axis has been shown to be involved in tight junction (TJ) regulation. H. pylori (Hp) infects the stomach of over half of the human population, causing a wide variety of disorders ranging from asymptomatic gastritis, to gastroduodenal ulcers and adenocarcinomas. The ability of certain strains of Hp to subvert normal cellular pathways regulating TJ permeability is believed to influence the clinical outcome of infection. Nevertheless, the mechanisms implicated in Hp-induced junctional disruptions of the gastric epithelium remain incompletely understood.
AIM: The aim of this study was to investigate the role of ROCK in Hp-induced TJ disruption in vitro, using a human gastric epithelial cell line (HGE-20).
METHODS: Confluent HGE-20 monolayers were infected with the Hp strains 60190 or SS1, at a multiplicity of infection of 100:1, for a period of 2-18 h. Samples were then collected for immunocytochemistry and microscopy, western blotting, and small Rho GTPase and ROCK activity assays.
RESULTS: Immunocytochemistry and western blotting demonstrated membrane-to cytosol translocation of claudin-4 in infected HGE-20 monolayers as early as 6h post-infection. A 150% increase in ROCK activity was observed at the same time point, and the use of the selective ROCK inhibitor Y27632 (10µM) 1h prior to bacterial challenge reduced loss of claudin-4 from the membrane. Furthermore, the treatment of HGE-20 monolayers with filter-sterilized conditioned media prepared from infected cell supernatant did not induce the typical Hp-induced increase in ROCK activity. Finally, the activity level of the small Rho GTPases Rac-1 and RhoA was significantly increased either 2 or 6 h post-infection, consistent with their involvement in the signaling events leading to ROCK activation and claudin-4 redistribution.
CONCLUSION: In conclusion, these findings demonstrate that the mechanism whereby Hp disrupts gastric epithelial barrier structure involves membrane-to-cytosol translocation of claudin-4. This effect appears to be mediated, at least in part, by small Rho GTPases and ROCK, independently of an epithelial or bacterial secreted factor.