NADPH Oxidase NOX5-S Research Articles

M1965 Phosphatidylinositol-Specific Phospholipase C (PI-PLC) Mediates Bile Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Esophageal Adenocarcinoma Cells

We have shown that NADPH oxidase NOX5-S (S = short, i.e. lacking EF-hand motifs at its N-terminal) is overexpressed in SEG1 esophageal adenocarcinoma cells associated with Barrett's esophagus (BE) and in BE with high-grade dysplasia. NOX5-S also contributes to increased cell proliferation and decreased apoptosis in SEG1 cells. We have also shown that low dose of bile acid increases cell proliferation through upregulation of NOX5-S. In the present study we examined the role of PI-PLC in bile acid-induced NOX5-S expression in SEG1-esophageal adenocarcinoma cells. In SEG1 cells, low dose of taurodeoxycholic acid (TDCA 10-11M, 24h) significantly increased NOX5-S expression and hydrogen peroxide (H2O2) production. TDCA-induced increase in NOX5-S expression and H2O2 production was significantly reduced by the PI-PLC inhibitor U73122, and the MAP kinase kinase inhibitor PD98059. In SEG1 cells transfected with NOX5-S luciferase reporter plasmid NOX5LP containing NOX5-S promoter fragment (-2501 to -1 from ATG), TDCA significantly increased the luciferase activity, an increase which was significantly decreased by U73122. In addition, TDCA significantly increased ERK2 phosporylation, which was significantly reduced by U73122. The data suggest that TDCA-induced NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Isoforms of PI-PLC β1, β3, γ1 and γ2 were detectable by Western blot analysis, but not β2, β4, δ1 and δ2. Knockdown of PI-PLC γ2 with siRNA significantly decreased TDCA-induced NOX5-S protein expression and H2O2 production. The data suggest that PI-PLC γ2 may be involved in TDCA-induced NOX5-S expression. We conclude that in SEG1 cells low dose of TDCA increases NOX5S expression and H2O2 production. This increase in NOX5-S expression may depend on sequential activation of PI-PLC and ERK MAP kinases. Supported by NIDDK R21 DK073327-01.

M1966 NADPH Oxidase NOX5-S Mediates Cyclin D1 Expression in Barrett's Esophageal Adenocarcinoma Cells

M1966 NADPH Oxidase NOX5-S Mediates Cyclin D1 Expression in Barrett's Esophageal Adenocarcinoma Cells

STAT5 mediates PAF-induced NADPH oxidase NOX5-S expression in Barrett's esophageal adenocarcinoma cells

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368-20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H(2)O(2)) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H(2)O(2) production. PAF-dependent induction of NOX5-S expression and H(2)O(2) production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA(2), and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA(2), whereas PAF-induced cPLA(2) phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H(2)O(2) production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA(2), and STAT5 in these cells.

NADPH Oxidase NOX5-S Mediates Acid-induced Cyclooxygenase-2 Expression via Activation of NF-κBin 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 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-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-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-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.

CAMP-response Element-binding Protein Mediates Acid-induced NADPH Oxidase NOX5-S Expression in Barrett Esophageal Adenocarcinoma Cells

Gastroesophageal reflux disease complicated by Barrett 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 known. We found that NOX1 and NOX5-S were the major isoforms of NADPH oxidase in SEG1-EA cells. The expression of NOX5-S mRNA was significantly higher in these cells than in esophageal squamous epithelial cells. NOX5 mRNA was also significantly higher in Barrett tissues with high grade dysplasia than without dysplasia. Pulsed acid treatment significantly increased H(2)O(2) production in both SEG1-EA cells and BE mucosa, which was blocked by the NADPH oxidase inhibitor apocynin. In SEG1 cells, acid treatment increased mRNA expression of NOX5-S, but not NOX1, and knockdown of NOX5 by NOX5 small interfering RNA abolished acid-induced H(2)O(2) production. In addition, acid treatment increased intracellular Ca(2+) and phosphorylation of cAMP-response element-binding protein (CREB). Acid-induced NOX5-S expression and H(2)O(2) production were significantly inhibited by removal of extracellular Ca(2+) and by knockdown of CREB using CREB small interfering RNA. Two novel CREB-binding elements TGACGAGA and TGACGCTG were identified in the NOX5-S gene promoter. Overexpression of CREB significantly increased NOX5-S promoter activity. Knockdown of NOX5 significantly decreased [(3)H]thymidine incorporation, which was restored by 10(-13) M H(2)O(2). Knockdown of NOX5 also significantly decreased retinoblastoma protein phosphorylation and increased cell apoptosis and caspase-9 expression. In conclusion, in SEG1 EA cells NOX5-S is overexpressed and mediates acid-induced H(2)O(2) production. Acid-induced NOX5-S expression depends on an increase in intracellular Ca(2+) and activation of CREB. NOX5-S contributes to increased cell proliferation and decreased apoptosis.