The Pathogenesis of Barrett’S Esophagus: the Combination of Acid and Bile Is Synergistic in the Induction of Cdx2 and Egfr Activation in Esophageal Cells
Nelly E. Avissar*, Liana Toia, Yingchuan Hu, Alexi Matousek, Daniel Raymond, Carolyn E. Jones, Thomas J. Watson, Jeffrey H. Peters
Surgery, University of Rocehster, School of Medicine and Dentistry, Rcohester, NY
Clinical evidence strongly suggests that mixed reflux of duodenal and gastric content is associated with esophageal mucosal injury, particularly the development of Barrett’s esophagus. Caudal homeobox 2 (CDX2), a transcription factor involved in normal intestinal development is thought to be the key factor responsible for the intestinal phenotype. We have previously shown that deoxycholic acid (DCA), at neutral pH induces CDX2 through transactivation of the epidermal growth factor receptor (EGFR). The aim of this study was to test the hypothesis that a combination of bile and acid pH is more potent in inducing CDX2 and activating the EGFR than either component alone.
Methods: SEG-1 human esophageal adenocarcinoma cells were incubated with acid alone (pH 5), deoxycholic acid alone (100 or 300 μM), or their combination for up to 24 hours. CDX2 mRNA was determined by real-time PCR and EGFR site-specific tyrosine phosphorylation and receptor degradation were determined by Western blot analysis.
Results: Neither acid (pH 5) nor the 100 μM DCA dose alone induced CDX2 mRNA. In contrast, there was a synergistic 55 fold increase in CDX2 mRNA expression with exposure to 100 μM DCA at pH5. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested, but in different time courses. Acid alone induced peak EGFR phosphorylation at 8h, DCA alone at 30 min and 8 hours and acid plus DCA at 1 hour. Interestingly, degradation of the EGF receptor occurred with the combination treatment but not with either acid or DCA alone, indicating differential transactivation pathways. The combination of pH 5 and 300 μM DCA resulted in significant cell death at all time points.
Conclusion: The addition of acid markedly enhances bile salt induced activation of the transcription factor CDX2 and occurs coincident with activation of the epidermal growth factor receptor. CDX2 gene induction occurs at significantly lower concentrations of bile salt than in the absence of acid, and may be due to differential EGFR transactivation. This data provides further insight into the molecular pathogenesis of Barrett’s columnar metaplasia and identifies molecular targets useful for diagnosis, risk assessment and therapeutic intervention.