Volume 121, Issue 6, Pages 1391-1399 (December 2001) Duodenal reflux induces cyclooxygenase-2 in the esophageal mucosa of rats: Evidence for involvement of bile acids  Fan Zhang, Nasser K. Altorki, Yu-Chung Wu, Robert A. Soslow, Kotha Subbaramaiah, Andrew J. Dannenberg  Gastroenterology  Volume 121, Issue 6, Pages 1391-1399 (December 2001) DOI: 10.1053/gast.2001.29781 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 CD and GCD induce PGE2 synthesis. SCC450 cells were treated with (A) 0–300 μmol/L CD or (B) 0–1500 μmol/L GCD for 6 hours. The medium was then replaced with DMEM/F12 containing 1% FCS and 10 μmol/L sodium arachidonate. Thirty minutes later, the medium was collected to determine the amount of PGE2 synthesis. (C) Cells were treated with vehicle, 300 μmol/L CD, or 300 μmol/L CD plus 0.1–10 μmol/L NS398 for 6 hours. After the addition of 10 μmol/L sodium arachidonate for 30 minutes, the medium was collected to determine amounts of PGE2. Production of PGE2 was determined by enzyme immunoassay. Columns, means; bars, SD. n = 4. *P < 0.05; **P < 0.01. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 CD is a more potent inducer of COX-2 than either GCD or TCD. SCC450 cells were treated for 6 hours with (A) 0–300 μmol/L CD; (B) 0–1250 μmol/L GCD or 200 μmol/L CD; and (C) 0–1000 μmol/L TCD, 0–1000 μmol/L GCD, 100 μmol/L CD, or 50 ng/mL PMA. (D) Human esophageal squamous carcinoma cell lines (30, 150, 170, 410, 1190) were treated for 6 hours with vehicle, 200 μmol/L CD, or 50 ng/mL PMA. Cellular lysate protein (30 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antiserum specific for COX-2. Ovine COX-2 was used as a standard. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 CD and GCD induce COX-2 mRNA. SCC450 cells were treated with vehicle, GCD (500–1250 μmol/L), or CD (25–200 μmol/L) for 3 hours. Total cellular RNA was isolated and 10 μg of RNA was added to each lane. The Northern blot was hybridized sequentially with probes that recognized COX-2 mRNA and 18S ribosomal RNA, respectively. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Acidifying the culture medium sensitizes esophageal cells to bile acid–mediated induction of COX-2. SCC450 cells were treated for 6 hours with vehicle, (A) 50–75 μmol/L CD, or (B) 250–750 μmol/L GCD at pH 6.5 or 7.3. Cellular lysate protein (30 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antiserum specific for COX-2. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Induction of COX-2 by CD is mediated by PI-3K. SCC 450 cells were treated for 6 hours with vehicle, 200 μmol/L CD, CD plus 1–10 μmol/L LY294002, or CD plus 0.1–1 μmol/L wortmannin. Cellular lysate protein (30 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was probed with antibody specific for COX-2. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 ERK1/2 MAPK is involved in bile acid–mediated induction of COX-2. (A) SCC 450 cells were treated with vehicle, PMA (50 ng/mL), or CD (200 μmol/L) for the indicated periods of time. Phospho-ERK1/2 was immunoprecipitated from equal amounts of cell lysates with anti–phospho-ERK1/2 antibody. Subsequently, ERK1/2 activity was measured by incubating the immunoprecipitate with ATP and Elk1 fusion protein as substrate. After electrophoresis, the immunoblot was probed with antibody to phospho-Elk1. (B) SCC 450 cells were treated with vehicle, 200 μmol/L CD, or CD plus 1–50 μmol/L PD98059 for 6 hours. Cellular lysate protein (30 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was probed with an antibody specific for COX-2. Ovine COX-2 was used as a standard. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 COX-2 is induced in the esophageal mucosa of rats with esophagoduodenal anastomoses. (A) Surgical esophagoduodenal anastomoses were created in 5 rats to induce reflux esophagitis (OP). These rats were killed 16 weeks later. Esophageal mucosa was obtained a minimum of 1.5 cm from the anastomotic site from control and OP rats. Tissue lysates were prepared for immunoblot analysis. (B) Western blot analysis of COX-2 was performed in esophageal (Es), stomach (St), and duodenal (Du) mucosal lysates in the same rats that had esophagoduodenal anastomoses used in A. In A and B, tissue lysate protein (100 μg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibody specific for COX-2. Ovine COX-2 was used as a standard. COX-2 expression was only detected in the esophageal mucosa of animals with esophagoduodenal anastomoses. Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 H&E and immunostaining for Ki-67 and cyclin D1 in rat esophageal tissues. (A, C, and E) Normal rat esophagus; (B, D, and F) esophagus from rats in which an esophagoduodenal anastomosis was created 16 weeks earlier. (A) The normal esophagus is lined by a relatively thin, keratinized, stratified squamous epithelium. In the normal state, the esophageal mucosa does not show marked basilar proliferation, elongated rete pegs, or significant inflammatory infiltrates (H&E, 40×). (B) The esophageal mucosa is markedly thickened and inflamed. The thickening is caused by a marked expansion of the basilar zone with consequent elongation of rete pegs. These features reflect reflux esophagitis (H&E, 40×). (C) The normal esophagus exhibits a thin basilar, proliferative zone, highlighted with the Ki-67 antibody that recognizes cells that have exited G0, the resting phase of the cell cycle (40×). (D) The expanded basilar proliferative zone of reflux esophagitis is characterized by a significantly increased component of Ki-67–reactive cells compared with normals (40×). (E) The normal esophagus is nearly devoid of cells expressing cyclin D1. Of note, the basilar proliferative zone is negative for cyclin D1 (40×). (F) In contrast, the expanded basilar proliferative zone of reflux esophagitis is characterized by a significantly increased component of cyclin D1–reactive cells. Comparison with sections evaluated with Ki-67 indicates that the proliferative basilar zone in reflux esophagitis coexpresses Ki-67 and cyclin D1 (100×). Gastroenterology 2001 121, 1391-1399DOI: (10.1053/gast.2001.29781) Copyright © 2001 American Gastroenterological Association Terms and Conditions