578 MEK1 Kinase and Rho Kinase ROCK2 Mediate Acid-Induced NOX5-S Expression and H 2O 2 Production in Barrett's Esophageal Adenocarcinoma Cells

Abstract

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We therefore investigated the role of NADPH oxidases in acid-induced changes in Barrett's EA cell line FLO. RT-PCR and 5'-RACE showed that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression was significantly greater in EA cell lines (FLO, and OE33) than in esophageal squamous epithelial cell line HET-1A. The levels of NOX5 mRNA were also markedly increased in human Barrett's mucosal biopsies with moderate dysplasia and in EA tissues, when compared with normal esophageal mucosa or BE mucosa. Immunohistochemical studies showed that NOX5-S was present in the cytosol of FLO cells. This result was further confirmed by transfection of FLO cells with GFP-tagged NOX5-S plasmid. In FLOEA cells, knockdown of NOX5-S by NOX5 siRNA significantly decreased cell proliferation at basal condition and inhibited acid-induced increase in cell proliferation, suggesting that NOX5-S may play a role in growth and proliferation of EA cells. Acid treatment significantly increased H2O2 production in both FLO cells and BE mucosa. Acid treatment significantly increased NOX5-S mRNA expression and H2O2 production, and knockdown of NOX5-S with NOX5 siRNA abolished acid-induced H2O2 production in FLO cells, indicating that NOX5-S mediates acid-induced H2O2 production. Acid-induced NOX5-S expression and H2O2 production were significantly decreased by knockdown of Rho kinase ROCK2 and MEK1 kinase with their siRNAs. Overexpression of constitutively active ROCK2 Rho kinase and constitutively active MEK1 kinase significantly increased NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased Rho kinase activity, an increase which was not affected by knockdown of MEK1 kinase. Overexpression of constitutively active ROCK2 significantly increased MEK1 phosphorylation. These data suggest that acidinduced NOX5-S expression depends on sequential activation of Rho kinase ROCK2 and MEK1 kinase. In conclusion, in EA cells acid induces H2O2 production by activation of NADPH oxidase NOX5-S and causes upregulation of NOX5-S expression through sequential activation of Rho kinase ROCK2 and MEK1 kinase. In these cells NOX5-S contributes to increased cell proliferation. Supported by NIH NIDDK R21 DK073327-01.