Protein concentration of the supernatant was determined using the BCA protein assay kit (Pierce, Rockford, IL). 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 occurs at the level of transcription, and involves activation of the PI3K/AKT/AP-1 pathway. and are the main mucin genes expressed in the stratified squamous epithelium, whereas is usually expressed in the submucosal glands 13. The gene is usually expressed in the stomach and in tracheobronchial cells, but not in the normal esophagus13, 14. De novo expression of the and the genes (an intestinal mucin) has been observed in Barretts esophagus 13, 15C17. 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 comprehended. 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) STING agonist-4 and unconjugated (CD and DC) bile acids were obtained 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 obtained from Novocastra Laboratories Ltd (Newcastle, UK). Mouse anti-MUC5AC antibody (clone SPM488) for rat tissues was obtained from Spring Bioscience (Fremont, CA). MUC5AC polyclonal antibody HO8 was a gift from Dr. Christopher M. Evans at The University of Texas M. D. Anderson Cancer Center. Cell Culture Human SKGT-4 EA cells, derived from well-differentiated adenocarcinoma arising in Barretts esophagus,18 were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 l/mL streptomycin at 37C in a humidified atmosphere of 95% air and 5% CO2. Treatments with vehicle (0.1% ethanol) or bile acids were performed in 0.5% FBS. Cytotoxicity was assessed by cell numbers, trypan blue exclusion, and the MTT assay STING agonist-4 (Promega, Madison, WI). For the trypan blue analysis, after treatment with bile acids for 16 hours, cells were mixed 1:1 with 0.4% trypan blue and examined for dye exclusion. Protein Isolation and Immunoblot Analysis Cells were lysed in buffer made up of 30 mM Tris-HCl (pH 6.8), 150 mM NaCl, 2 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 2 mM orthovanadate, 1% Triton X-100, 1% NP-40, 0.2 mM phenylmethylsulfonyl fluoride, and one mini-tablet protease inhibitor cocktail (Roche Diagnostics Corp, Indianapolis, IN). Protein concentration of the supernatant was decided using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were subjected to electrophoresis on either 3C8% Tris-acetate gradient gels for MUC5AC detection or 10% Tris-glycine gels for detection of other proteins. After gel electrophoresis and transfer to nitrocellulose, the membranes were stained with 0.5% Ponceau S with 1% acetic acid to confirm equal loading and transfer efficiency. Western blots were performed by incubating membranes at room temperature for 1 hour in a blocking solution made up of 5% nonfat dry milk and 0.1% Tween-20 in Tris-buffered saline (10 mM Tris-HCl with 150 mM NaCl, pH Igfbp3 7.6), probing with specific primary antibodies, washing with Tris-buffered saline containing 0.1% Tween 20, and probing with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were visualized by chemiluminescence.. Plasmids, Transient Transfection, and Luciferase Reporter Assays A human MUC5AC promoter construct19, made up of the 3.7 kb MUC5AC 5-flanking region fused to a luciferase reporter gene was provided by Dr. Ja Seok Koo at the University of Texas M. D Anderson Cancer Center (MDACC). Dominant-negative AP-1 (blunted TAM67) 20 and the AP-1 promoter-luciferase reporter 21 were provided by Dr. Shrikanth A. Reddy and Dr. Jonathan Kurie (MDACC). The cDNA plasmid for the dominant-negative mutant of AKT (AKT AAA) was STING agonist-4 provided by Dr. Dimpy Koul.