Helicobacter pylori-activated gastric fibroblasts induce epithelial-mesenchymal transition of gastric epithelial cells in vitro in a TGF-β-dependent manner

Background Colonization of the gastric mucosa with Helicobacter pylori (Hp) leads to the cascade of pathologic events including local inflammation, gastric ulceration, and adenocarcinoma formation. Paracrine loops between tissue cells and Hp contribute to the formation of gastric cancerous loci; however, the specific mechanisms underlying existence of these loops remain unknown. We determined the phenotypic properties of gastric fibroblasts exposed to Hp (cagA+vacA+) infection and their influence on normal epithelial RGM-1 cells. Materials and Methods RGM-1 cells were cultured in the media conditioned with Hp-activated gastric fibroblasts. Their morphology and phenotypical changes associated with epithelial-mesenchymal transition (EMT) were assessed by Nomarski and fluorescence microscopy and Western blot analysis. Motility pattern of RGM-1 cells was examined by time-lapse video microscopy and transwell migration assay. The content of TGF-beta in Hp-activated fibroblast-conditioned media was determined by ELISA. Results The supernatant from Hp-activated gastric fibroblasts caused the EMT-like phenotypic diversification of RGM-1 cells. The formation of fibroblastoid cell sub-populations, the disappearance of their collective migration, an increase in transmigration potential with downregulation of E-cadherin and upregulation of N-cadherin proteins, prominent stress fibers, and decreased proliferation were observed. The fibroblast (CAF)-like transition was manifested by increased secretome TGF-beta level, alpha-SMA protein expression, and its incorporation into stress fibers, and the TGF-beta R1 kinase inhibitor reduced the rise in Snail, Twist, and E-cadherin mRNA and increased E-cadherin expression induced by CAFs. Conclusion Gastric fibroblasts which are one of the main targets for Hp infection contribute to the paracrine interactions between Hp, gastric fibroblasts, and epithelial cells. TGF-beta secreted by Hp-activated gastric fibroblasts prompting their differentiation toward CAF-like phenotype promotes the EMT-related phenotypic shifts in normal gastric epithelial cell populations. This mechanism may serve as the prerequisite for GC development.