NADPH Oxidase NOX5-S Mediates Acid-induced Cyclooxygenase-2 Expression via Activation of NF-κBin Barrett's Esophageal Adenocarcinoma Cells*

Abstract

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 E₂ (PGE₂) 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 PGE₂ production. Knockdown of NOX5-S or NF-κB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE₂ production in SEG1 cells. Acid treatment significantly decreased IκBα and increased luciferase activity when SEG1 cells were transfected with an NF-κB in vivo activation reporter plasmid, pNF-κB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IκBα was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-κB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H₂O₂ production, COX2 expression, PGE₂ 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 PGE₂ production depend on an increase in cytosolic Ca²⁺ and sequential activation of NOX5-S and NF-κB in SEG1 cells. COX2-derived PGE₂ production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.