Volume 122, Issue 2, Pages 299-307 (February 2002) Acid exposure activates the mitogen-activated protein kinase pathways in Barrett's esophagus  Rhonda F. Souza, Kenneth Shewmake, Lance S. Terada, Stuart Jon Spechler  Gastroenterology  Volume 122, Issue 2, Pages 299-307 (February 2002) DOI: 10.1053/gast.2002.30993 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Acid caused an immediate increase in ERK activity. SEG-1 cells were exposed to acid for 3 minutes, and ERK activity (measured as Elk-1 phosphorylation) was determined at the indicated times. Fetal bovine serum (FBS) was used as a positive control. A representative experiment demonstrating (A) Elk-1 phosphorylation and (B) Western blot for total ERK is shown. (C) ERK activity increased immediately after acid exposure (*P < 0.05). The histogram shows the mean ± SEM of 3 individual determinations. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Acid caused an immediate increase in p38 activity. SEG-1 cells were exposed to acid for 3 minutes, and p38 activity (measured as ATF-2 phosphorylation) was determined at the indicated times. Fetal bovine serum (FBS) was used as a positive control. A representative experiment demonstrating (A) ATF-2 phosphorylation and (B) Western blot for total p38 is shown. (C) p38 activity increased immediately and remained significantly elevated 3 and 5 minutes after acid exposure (*P < 0.05). The histogram shows the mean ± SEM of 3 individual determinations. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Acid caused a delayed increase in JNK activity. SEG-1 cells were exposed to acid for 3 minutes, and JNK activity (measured as c-Jun phosphorylation) was determined at the indicated times. Fetal bovine serum (FBS) was used as a positive control. A representative experiment demonstrating (A) c-Jun phosphorylation and (B) Western blot for total JNK is shown. (C) JNK activity increased significantly 3 minutes after acid exposure (*P < 0.05). The histogram shows the mean ± SEM of 3 individual determinations. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Acid increased SEG-1 cell numbers. SEG-1 cells were exposed to acid for 3 minutes, and cell counts were determined using a Coulter counter at 24 hours. SEG-1 cells not exposed to acid served as corresponding controls. Acid increased cell number compared with untreated control cells. *P < 0.05. The histogram shows the mean ± SEM of 6 individual determinations. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Acid increased cell survival and stimulated cell-cycle entry in SEG-1 cells. SEG-1 cells were exposed to acid alone, acid in the presence of the ERK inhibitor PD98059, acid in the presence of the p38 inhibitor SB203580, or acid in the presence of dominant-negative JNK1/2 mutants; flow cytometric analysis was performed after 3 hours (for subG0 and G0/G1) and after 24 hours (for S and G2/M). SEG-1 cells not exposed to acid served as the corresponding controls. The histograms indicate the percentage of cells in each phase of the cell cycle. (A) The subG0 fraction decreased significantly in cells treated with acid alone, acid in the presence of SB203580, and acid in the presence of dominant-negative JNK1/2 mutants compared with corresponding controls. (B) The G0/G1 fraction increased in cells treated with acid alone, acid in the presence of SB203580, and acid in the presence of dominant-negative JNK1/2 mutants compared with corresponding controls. (C) The S fraction did not change with acid exposure. (D) The G2/M fraction increased in cells treated with acid alone and with acid in the presence of PD98059 compared with corresponding controls. These findings suggest that acid activation of ERK increases cell survival, whereas acid activation of p38 and JNK promotes entry into the cell cycle. *P < 0.05 compared with corresponding non–acid-exposed control cells. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Inhibition of MAPKs blocked the acid-induced increase in SEG-1 cell numbers. SEG-1 cells were exposed to acid alone, acid in the presence of the ERK inhibitor PD98059, or acid in the presence of the p38 inhibitor SB203580. Cell counts were determined using a Coulter counter at 24 hours. SEG-1 cells not exposed to acid served as corresponding controls. Acid alone significantly increased cell counts; treatment with PD98059 or SB203580 blocked the acid-induced increase in cell number. *P < 0.05 compared with non–acid-exposed corresponding control cells. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Acid activation of MAPKs decreased apoptosis in SEG-1 cells. SEG-1 cells were exposed to acid alone, acid in the presence of the ERK inhibitor PD98059, or acid in the presence of the p38 inhibitor SB203580. Apoptosis was determined using an enzyme-linked immunosorbent assay at 24 hours. SEG-1 cells not exposed to acid served as corresponding controls. Acid alone significantly decreased apoptosis; treatment with PD98059 or SB203580 blocked the acid-induced decrease in apoptosis. Data are expressed as the ratio of absorbance (cells exposed to acid alone or in the presence of PD98059 or SB203580) to absorbance (non–acid-exposed corresponding control cells). *P < 0.05 compared with non–cid-exposed corresponding control cells. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Acid increased p38 activity in the metaplastic mucosa of patients with BE. The esophagus of 7 patients with Barrett's mucosa was perfused with acid for 3 minutes, and MAPK activities (measured as ATF-2, Elk-1, or JNK-1 phosphorylation) were determined in esophageal biopsy specimens immediately after acid exposure. (A) Representative experiment showing ATF-2, Elk-1, and JNK-1 phosphorylation. (B) p38 activity increased significantly after acid exposure (P < 0.05). Scatter plots show MAPK activity in the metaplastic mucosa before and after acid perfusion. The dotted line indicates the mean value for each group. Gastroenterology 2002 122, 299-307DOI: (10.1053/gast.2002.30993) Copyright © 2002 American Gastroenterological Association Terms and Conditions