Immunohistochemistry studies were performed on formalin-fixed, paraffin-embedded sections (6 m) with antibodies against MUC5AC antibodies (mouse monoclonal antibody; dilution 1:100; HO8 polyclonal antibody, dilution 1:200) after antigens were retrieved using 10 mM sodium citrate buffer as previously described26. == Results == == MUC5AC Mucin Expression in Human Esophageal Mucosa, Barretts Esophagus, and Esophageal Adenocarcinoma == To clarify the pattern of MUC5AC expression in normal human esophageal mucosa, Barretts esophagus, and esophageal adenocarcinoma, we performed immunohistochemistry to detect MUC5AC apomucin in paired human tissue specimens. bile acids than by unconjugated bile acids, and this occurred at the transcriptional level. In the rat reflux model, MUC5AC mucin was abundantly expressed in tissues of BE stimulatesd by duodenoesophageal reflux. Conjugated bile acids induced AKT phosphorylation in SKGT-4 cells, but had no effects on ERK1/2, JNK, and P-38 kinase phosphorylation. The PI3K inhibitor (Z)-SMI-4a LY294002 and a dominant-negative AKT construct prevented the induction of MUC5AC by conjugated bile acids. Transactivation of AP-1 by conjugated bile acids coincided with MUC5AC induction, and co-transfection with a dominant-negative AP-1 vector decreased MUC5AC transcription and its induction. == Conclusions == Conjugated bile acids in the bile refluxate contribute to MUC5AC induction in the esophagus. This (Z)-SMI-4a occurs at the level of transcription, (Z)-SMI-4a and involves activation of the PI3K/AKT/AP-1 pathway. Keywords:MUC5AC mucin, bile reflux, PI3K pathway, AP-1, Barretts Esophagus == Introduction == Esophageal cancer is (Z)-SMI-4a the seventh leading cause of cancer death among men in the U.S1. During the past several decades, the incidence of adenocarcinoma in the esophagus has risen at an alarming rate in western countries, and has exceeded that of squamous cell carcinoma2,3. Most esophageal adenocarcinomas (EA) arise from Barretts esophagus4, a condition in which metaplastic columnar epithelium replaces the normal stratified squamous epithelium.5 Gastroesophageal reflux of acid and bile is the predominant initiating factor in Barretts metaplasia and its progression to EA6. Bile reflux is particularly common in individuals with gastroesophageal reflux disease who subsequently develop Barretts esophagus7,8. Barretts esophagus also develops secondary to bile reflux in patients who have undergone total gastrectomy9. Consistent with this observation, Barretts esophagus is followed by EA in a rat model that uses esophagojejunostomy to bypass exposure to acid reflux from the stomach10. In this model, enteroesophageal reflux produces EA in 48% of rats in the absence of exposure to any exogenous carcinogen11. The precise mechanisms by which duodenal reflux causes esophageal injury and leads to metaplasia and predisposes to EA is uncertain. Mucins, are large, heavily glycosylated proteins located at the surface of many epithelia and play an important role in protecting epithelial cells12. In normal esophageal tissue, secreted mucins protect the mucosa against potential injuries such as the reflux of gastroduodenal contents, including acid and bile4. Mucin genes are expressed in a site-specific manner throughout the gastrointestinal tract. In the normal esophagus,MUC1andMUC4are the main mucin genes expressed in the stratified squamous epithelium, whereasMUC5Bis expressed in the submucosal glands13. TheMUC5ACgene is expressed in the stomach and in tracheobronchial cells, but not in the normal esophagus13,14. De novo expression of theMUC5ACand theMUC2genes (an intestinal mucin) has been observed in Barretts esophagus13,1517. The associated risk factors, the mechanisms controlling the expression of MUC5AC mucin in Barretts esophagus, and the pathological significance of this mucin in Barretts columnar epithelium are not clearly understood. We hypothesized that bile acids in the gastroesophageal refluxate contribute to the formation of Barretts phenotype, including the ectopic expression of MUC5AC mucin. We sought to determine which bile acids are responsible for MUC5AC expression and the molecular mechanisms involved. == Materials and Methods == == Materials == Dulbeccos modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies, Inc. (Grand Island, NY). LY294002 was purchased from Calbiochem (San Diego, CA). Conjugated (GC, TC, TCDC, GCDC, TDC) and unconjugated (CD and DC) bile acids were from Sigma (St. Louis, MO). ECL-chemiluminescence reagents were purchased from Amersham-Pharmacia (Piscataway, NJ). Total and phosphorylated ERK-1/2 antibodies, phosphorylated AKT antibodies, and phosphorylated JNK and P-38 antibodies were purchased from Cell Signaling Technology (Beverly, MA). MUC5AC monoclonal antibody (CLH2 clone) was from Novocastra Laboratories Ltd (Newcastle, UK). Mouse anti-MUC5AC antibody (clone SPM488) for rat cells was from Spring Bioscience (Fremont, CA). MUC5AC polyclonal antibody HO8 was a gift from Dr. Christopher M. Evans in the University of Texas M. D. Anderson Malignancy Center. == Cell Tradition == Human being SKGT-4 EA Mouse monoclonal to MUM1 cells, derived from well-differentiated adenocarcinoma arising in Barretts esophagus,18were managed in DMEM supplemented.