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

Hong J, Li D, Cao W - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America.

ABSTRACT
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.

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Role of calcium in Rho kinase activation.A. Acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, suggesting that acid-induced activation of Rho kinase may be dependent on intracellular calcium increase. B) A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. N = 3, ANOVA * P<0.05, compared with control group; # P<0.05, compared with acid group; t test ** P<0.05.
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pone.0149735.g006: Role of calcium in Rho kinase activation.A. Acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, suggesting that acid-induced activation of Rho kinase may be dependent on intracellular calcium increase. B) A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. N = 3, ANOVA * P<0.05, compared with control group; # P<0.05, compared with acid group; t test ** P<0.05.

Mentions: We found that acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity (Fig 6A) in FLO-1 cells and NOX5-S mRNA expression in FLO-1 (Fig 7A) and OE33 cells (Fig 7C), an increase which was blocked by the removal of calcium, suggesting that acid-induced activation of Rho kinase and increase in NOX5-S expression may be dependent on the intracellular calcium increase. To further confirm these results, we exposed FLO-1 and OE33 cells to the calcium ionophore A23187. A23187 is a bacterially derived antibiotic calcium ionophore, which allows calcium ions to cross biological membranes [44]. Fig 6B shows that A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. Similarly, A23187 significantly increased NOX5-S mRNA expression in FLO-1 cells (Fig 7B) as well as in OE33 cells (Fig 7D).


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

Hong J, Li D, Cao W - PLoS ONE (2016)

Role of calcium in Rho kinase activation.A. Acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, suggesting that acid-induced activation of Rho kinase may be dependent on intracellular calcium increase. B) A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. N = 3, ANOVA * P<0.05, compared with control group; # P<0.05, compared with acid group; t test ** P<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764682&req=5

pone.0149735.g006: Role of calcium in Rho kinase activation.A. Acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, suggesting that acid-induced activation of Rho kinase may be dependent on intracellular calcium increase. B) A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. N = 3, ANOVA * P<0.05, compared with control group; # P<0.05, compared with acid group; t test ** P<0.05.
Mentions: We found that acid treatment (pH 4.0, 1 hour) significantly increased Rho kinase activity (Fig 6A) in FLO-1 cells and NOX5-S mRNA expression in FLO-1 (Fig 7A) and OE33 cells (Fig 7C), an increase which was blocked by the removal of calcium, suggesting that acid-induced activation of Rho kinase and increase in NOX5-S expression may be dependent on the intracellular calcium increase. To further confirm these results, we exposed FLO-1 and OE33 cells to the calcium ionophore A23187. A23187 is a bacterially derived antibiotic calcium ionophore, which allows calcium ions to cross biological membranes [44]. Fig 6B shows that A23187 significantly increased Rho kinase activity, suggesting that intracellular calcium increase may activate Rho kinase in FLO-1 EA cells. Similarly, A23187 significantly increased NOX5-S mRNA expression in FLO-1 cells (Fig 7B) as well as in OE33 cells (Fig 7D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus