NOX5-S Expression Research Articles

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

Tu1906 Direct Activation of MEK1 Kinase by Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Barrett's Associated Esophageal Adenocarcinoma Cells

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 have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Tu1911 A Novel Mechanism for Radiocontrast-Induced Pancreatic NF-KB Translocation and Injury Through Triggering Calcium and Calcineurin Activation

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 have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Tu1907 Curcumin Reduces the Inflammatory Factors of Reflux Esophagitis Through Non PKC Pathway in Part in Rat Model

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 have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.

Signaling in H2O2-Induced Increase in Cell Proliferation in Barrett's Esophageal Adenocarcinoma Cells

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have previously shown that NADPH oxidase NOX5-S generates reactive oxygen species (ROS) when Barrett's metaplastic cells are exposed to acid. Besides metaplastic cells, other H(2)O(2)-producing cells (e.g., inflammatory cells) present in BE mucosa may produce additional ROS, which may also affect metaplastic cells contributing to esophageal tumorigenesis. In this study, we investigate whether exogenous H(2)O(2) stimulates cell proliferation by increasing NOX5-S expression. Low dose (10(-13) M) of H(2)O(2) significantly increased thymidine incorporation, NOX5-S mRNA, and protein expression in a Barrett's EA cell line FLO. H(2)O(2)-induced increase in NOX5-S expression was significantly inhibited by knockdown of nuclear factor (NF)-κB1 p50 with p50 small interfering RNA (siRNA) in EA cell lines FLO and OE33. H(2)O(2) significantly increased p65 phosphorylation and the luciferase activity in FLO cells transfected with a NF-κB activation reporter plasmid pNF-κB-Luc. H(2)O(2)-induced increase in luciferase activity in FLO cells was significantly decreased by knockdown of extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase (MAPK). Overexpression of p50 and p65 remarkably increased the luciferase activity in FLO cells transfected with a NOX5-S reporter plasmid NOX5-LP. In addition, H(2)O(2)-induced thymidine incorporation in FLO cells was significantly decreased by the MAPK kinase 1/2 inhibitor 2'-amino-3'methoxyflavone (PD98059) and ERK2 siRNA but not by ERK1 siRNA. Likewise, H(2)O(2)-induced increase in NOX5-S expression was significantly decreased by ERK2 siRNA in FLO and OE33 cells. We conclude that a low dose of H(2)O(2) increases cell proliferation. H(2)O(2)-induced increase in cell proliferation may depend on sequential activation of ERK2 MAPK, NF-κB1 p50, and NOX5-S.

Bile Acid Reflux Contributes to Development of Esophageal Adenocarcinoma via Activation of Phosphatidylinositol-Specific Phospholipase Cγ2 and NADPH Oxidase NOX5-S

Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA.

Role of a novel bile acid receptor TGR5 in the development of oesophageal adenocarcinoma

Background and aimsMechanisms of the progression from Barrett's oesophagus to oesophageal adenocarcinoma (OA) are not fully understood. Bile acids may have an important role in this progression. This study aimed...

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

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.

582 Manometry and Impedance Characteristics of the Esophagus in Patients with Early Stage of Idiopathic Parkinson's Disease

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.

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.

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.

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.