NADPH oxidase NOX5-S mediates acid-induced cyclooxygenase-2 expression via activation of NF-κB in Barrett's esophageal adenocarcinoma cells

We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid- induced increase in prostaglandin E-2 (PGE(2)) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE(2) production. Knockdown of NOX5-S or NF-kappa B1 p50 by their small interfering RNA significantly inhibited acid- induced COX2 expression and PGE(2) production in SEG1 cells. Acid treatment significantly decreased I kappa B alpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappa B in vivo activation reporter plasmid, pNF-kappa B-Luc. In a novel Barrett's cell line overexpressing NOX5-S, I kappa B alpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappa B-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE(2) production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE(2) production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappa B in SEG1 cells. COX2-derived PGE(2) production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.