Bile acids directly augment caudal-related homeobox gene Cdx2 expression in oesophageal keratinocytes in Barrett’s epithelium Running title: Bile acids and Cdx2

Background & Aims: The mechanism of transformation to intestinal metaplasia in Barrett’s oesophagus has not been clarified. We investigated the effects of various bile acids on the expression of the caudal-related homeobox gene Cdx2 in cultured oesophageal squamous epithelial cells. In addition, morphological and histochemical changes of squamous cells to intestinal epithelial cells were studied in response to bile acid-induced expression of Cdx2. Methods: A rat model of Barrett’s oesophagus was created by anastomosing the oesophagus and jejunum, and Cdx2 expression was investigated by immunohistochemistry. Further, the response of various bile acids on Cdx2 gene expression was studied in human colon epithelial cell lines Caco-2 and HT-29, as well as cultured rat oesophageal squamous epithelial cells using a Cdx2 promoter luciferase assay. In addition, primary cultured oesophageal squamous epithelial cells were transfected with Cdx2 expression vectors and their possible transformation to intestinal type epithelial cells was investigated. Results: Esophago-jejunal anastomoses formed intestinal goblet cell metaplasia in rat oesophagus specimens and metaplastic epithelia strongly expressed Cdx2. When the effects of 11 kinds of bile acids on Cdx2 gene expression were examined, only cholic acid (CA) and dehydrocholic acid dose-dependently increased the Cdx2 promoter activity and Cdx2 protein production in Caco-2 and HT-29, and cultured rat oesophageal keratinocytes. Results from the mutation analysis of Cdx2 promoter suggested that 2 NF-κB binding sites are responsible for the bile acid-induced activation of the Cdx2 promoter. When bile acids were measured in the oesophageal refluxate of the rats with experimental Barrett’s oesophagus, the concentration of CA was found to be consistent with the experimental dose that augmented Cdx2 expression in vitro. Further, transfection of the Cdx2 expression vector in cultured rat oesophageal keratinocytes induced production of intestinal type mucin, MUC2, in cells that expressed Cdx2. Conclusions: We found that CA activates Cdx2 promoter via NF-κB and stimulates the production of Cdx2 protein in oesophageal keratinocytes with production of intestinal type mucin. This may be one of the mechanisms of metaplasia in Barrett’s oesophagus. group.bmj.com on October 30, 2017 Published by http://gut.bmj.com/ Downloaded from