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

Abstract

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.Esophageal adenocarcinoma has increased in incidence over the past 3 decades (1), at a rate exceeding that of any other cancer in the last 10 years (2, 3). Esophageal adenocarcinoma is characterized by a uniformly poor prognosis, with a median survival time following diagnosis of less than 18 months, and a 5-year survival rate of less than 20% in operable tumors (4). The major risk factor for esophageal adenocarcinoma is gastroesophageal reflux disease (GERD) 2 complicated by Barrett esophagus (BE) (5). Approximately 10% of GERD patients develop BE, where esophageal squamous epithelium damaged by acid reflux is replaced by a metaplastic, intestinal type epithelium. The specialized intestinal metaplasia of BE is associated with a 30 -125-fold increased risk for the development of esophageal adenocarcinoma, with the best estimates of cancer incidence of about 0.5-1.0% per year, i.e. one cancer per 100 -200 patients for each year of observation (6 -8). A middle-aged individual with BE for 20 years or more has an estimated 10 -20% lifetime risk of developing esophageal adenocarcinoma, which is similar to the risk of lung cancer among heavy smokers or of liver cancer amo...

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

“…We show in this study that at least one isoform of Nox5 is responsible for PDGF-induced HASMC proliferation. This finding is in concert with studies of Nox5 in several cancer cell lines [25][26][27] and in human microvascular endothelial cells [28]. Further work will be necessary to investigate the participation of Nox5 in other cellular functions and to characterize the exact role of each of its isoforms.…”

“…With regard to PDGF signaling, Nox5 is of particular interest because it is activated by calcium and produces ROS, both of which have been shown to be required for PDGF-induced proliferation. Nox5 has been detected in several cancer cell lines [25,26] and, in a study with prostate cancer cells, appears to regulate cell growth [27]. Furthermore, the knockdown of Nox5 in human endothelial cells prevents proliferation [28].…”

Oncogenic Ras upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphorylation of GATA-6

“…Cyclic AMP response element-binding proteins have been found to be responsible for acid-induced expression of Nox5-S in barrett esophagel adenocarcinoma cells (Fu et al, 2006). Our present studies define the essential regulatory role of GATA-6 in the transcription of Nox1 in colorectal epithelium.…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…In many of cases, the source of this ROS is Nox enzymes: this includes melanoma (Nox4, [127]), prostate cancer (Nox5 [128] and Nox1 [129]), glioblastoma (Nox4 and sometimes Nox5 [130]), H. pylorusinduced gastric inflammation leading to gastric cancer (Nox1 [131]) and Barrett's esophageal adenocarcinoma (Nox5 [132]). Some, but not all reports have observed an increase in Nox1 expression in colon cancer, see [133][134][135][136].…”

“…Decreasing the expression of Nox1 (originally called Mox1) decreased cell division in vascular smooth muscle [46] and in V12-K-Ras transformed NRK cells [137]. Suppression of Nox5 expression in Barrett's esophageal adenocarcinoma cells likewise inhibited proliferation [132]. While in vivo studies are needed to definitively link Nox-derived ROS to the cancer phenotype, Nox-dependent effects on cell division, angiogenesis, cell survival and integrin signaling provide plausible mechanisms by which Nox enzymes may be causally linked to cancer development, and justify interest in Nox enzymes as drug targets for cancer prevention and treatment.…”