H. pylori vaca cytotoxin interferes with the RHO/RAC-1 signal cascade in wounded gastric epithelial monolayers: A key to inhibition of reepithelialization

Abstract

Re-epithelialization is an essential process for repair of gastric mucosal injury and ulcer healing. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and interferes with EGF-activated signal transduction. Several members of the Rho subfamily of GTPbinding proteins, including Rat-1 and Rho, regulate a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesion which are essential for cell migration. The aims of this study were to determine: a) the effect of H. pylori VacA cytotoxin on levels of Rho and Rac-1 proteins, and ADP-ribosylation levels of Rho in gastric epithelial cells and b) the effect of VacA on reepitbelialization in a wounded gastric cells monolayer model. Methods: VacA purified from H. pylori 60190 or a corresponding VacA-negative preparation from 1-1. pylori 60190-vl (an isogenic mutant strain in which VacA gene has been disrupted) were used. Rat gastric epithelial cells (RGM1) were incubated with either medium only (DMEM/F-12 with 5% FBS), medium containing VacA or medium containing the VacA-negative preparation for 24 and 48 hours. Standard excisional wounds (30 ram) were made in gastric epithelial cells monolayers with a razor blade and treated with H. pylori VacA or VacA-negative preparation as above. Studies: A) Rho and Rac-1 proteins levels by Western blot analysis, B) Rho ribosylation levels by ADPribosylation assay using [32p] NAD with C3 exoenzyme. C) Localization of Rac-1 after immunostaining and D) Actin microfilament assembly using a video image system. Results: VacA (but not a VacA-negative prep) significantly inhibited RGM1 cell migration by 25% (p < 0.03), reduced Rac1 expression by 65% (p < 0.04) and reduced C3-mediated ADP-ribosylation of Rho by 21% (p < 0.03). VacA cytotoxin (but not the VacA-negative prep) inhibited assembly of actin stress fibers and reduced Rac-1 expression in migrating epithelial cells. C_onclusions: H. pylori VacA significantly: 1) inhibits re-epithelialization of wounded gastric epithelial monolayers, 2) reduces Rac-1 expression, 3) affects Rho -ribosylation and 4) interferes with actin filament assembly. These data provide molecular mechanisms for H. pylori cytotoxin interference with re-epithelialization.