NF-κB Mediates Acid-Induced mPGES1 Expression in Barrett's Esophageal Adenocarcinoma Cells
Weibiao Cao, Xiaoxu Zhou, Dan Li, Jose Behar, Jack Wands, Murray Resnick. Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI
Background: Cycloxygenase-2 (COX2)-derived prostaglandin E2 (PGE2) may play an important role in esophageal tumorigenesis. We have shown that COX2 mediates acid-induced increase in PGE2 production and cell proliferation and that NADPH oxidase NOX5-S mediates acid-induced up-regulation of COX2. Which PGE synthase (PGES) is responsible for acid-induced PGE2 production in Barrett's esophagus (BE), however, is unknown. In this study we examined the role of mPGES1 in the acid-induced increase in PGE2 production and cell proliferation, and studied the role of NF-κB in acid-induced upregulation of mPGES1 in an esophageal adenocarcinoma (EA) cell line FLO.
Design: PGES mRNAs were measured by real-time PCR. Transfection of NOX5 and p50 siRNAs and plasmids were carried out by using Lipofectamine 2000.
Results: RT-PCR showed that mPGES1, mPGES2 and cytosolic PGES (cPGES) were present in FLO cells. mPGES1 protein levels were significantly increased in EA cell lines FLO and OE33. Pulsed acid treatment increased mPGES1 mRNA by 110% and mPGES2 by 10%, but did not have any effect on cPGES mRNA. Acid treatment significantly increased mPGES1 protein expression. Knockdown of mPGES1 by mPGES1 siRNA blocked acid-induced increase in PGE2 production and thymidine incorporation (an indicator of cell proliferation rate). Knockdown of NOX5-S by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a NF-κB in vivo activation reporter plasmid pNF-κB-Luc. Knockdown of NF-κB1 p50 by p50 siRNA almost abolished acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. In a chromatin immunoprecipitation assay, the promoter region of mPGES1 DNA was detectable in the immunoprecipitated chromatin sample of FLO cell lysate with a p50 antibody, indicating that p50 binds to mPGES1 promoter. Overexpression of p50 and p65 significantly increased the luciferase activity in FLO cells transfected with a mPGES1 reporter plasmid.
Conclusions: We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA.
Supported by NIH NIDDK R01 DK080703.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 273, Wednesday Morning