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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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NOX5-S expression and H2O2 production in FLO-1 EA cells after ROCK1 overexpression.(A) A typical example of three Western blot analysis showed that ROCK1 protein was successfully overexpressed in FLO-1 EA cells after transfection with ROCK1 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of ROCK1 constitutively active protein had no significant effect on NOX5-S expression. (D) Overexpression of ROCK1 constitutively active protein did not significantly affect H2O2 production in FLO-1 cells. The data suggest that acid-induced increase in NOX5-S expression and H2O2 production may not depend on activation of ROCK1 in FLO-1 EA cells. N = 3, ANOVA, there is no significant difference among control plasmid group, ROCK1 wild type plasmid group and ROCK1 constitutively active plasmid group.
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pone.0149735.g005: NOX5-S expression and H2O2 production in FLO-1 EA cells after ROCK1 overexpression.(A) A typical example of three Western blot analysis showed that ROCK1 protein was successfully overexpressed in FLO-1 EA cells after transfection with ROCK1 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of ROCK1 constitutively active protein had no significant effect on NOX5-S expression. (D) Overexpression of ROCK1 constitutively active protein did not significantly affect H2O2 production in FLO-1 cells. The data suggest that acid-induced increase in NOX5-S expression and H2O2 production may not depend on activation of ROCK1 in FLO-1 EA cells. N = 3, ANOVA, there is no significant difference among control plasmid group, ROCK1 wild type plasmid group and ROCK1 constitutively active plasmid group.

Mentions: Next we transfected ROCK1 and ROCK2 expression plasmids into FLO-1 cells, respectively. Transfection of ROCK1 or ROCK2 plasmids remarkably increased ROCK1 or ROCK2 protein expression in FLO-1 cells, when compared with control (Fig 4A, S5 Fig and Fig 5A, S7 Fig), indicating that ROCK1 and ROCK2 proteins were successfully overexpressed in FLO-1 cells. Overexpression of the constitutively active ROCK2 significantly enhanced NOX5-S expression (Fig 4B and 4C, S6 Fig) and H2O2 production (Fig 4D) in FLO-1 EA cells, when compared with the control. However, overexpression of the constitutively active ROCK1 had no statistically significant effect on NOX5-S protein expression (Fig 5B and 5C, S8 Fig) and H2O2 production (Fig 5D) in FLO-1 EA cells, when compared with the control.


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)

NOX5-S expression and H2O2 production in FLO-1 EA cells after ROCK1 overexpression.(A) A typical example of three Western blot analysis showed that ROCK1 protein was successfully overexpressed in FLO-1 EA cells after transfection with ROCK1 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of ROCK1 constitutively active protein had no significant effect on NOX5-S expression. (D) Overexpression of ROCK1 constitutively active protein did not significantly affect H2O2 production in FLO-1 cells. The data suggest that acid-induced increase in NOX5-S expression and H2O2 production may not depend on activation of ROCK1 in FLO-1 EA cells. N = 3, ANOVA, there is no significant difference among control plasmid group, ROCK1 wild type plasmid group and ROCK1 constitutively active plasmid group.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0149735.g005: NOX5-S expression and H2O2 production in FLO-1 EA cells after ROCK1 overexpression.(A) A typical example of three Western blot analysis showed that ROCK1 protein was successfully overexpressed in FLO-1 EA cells after transfection with ROCK1 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of ROCK1 constitutively active protein had no significant effect on NOX5-S expression. (D) Overexpression of ROCK1 constitutively active protein did not significantly affect H2O2 production in FLO-1 cells. The data suggest that acid-induced increase in NOX5-S expression and H2O2 production may not depend on activation of ROCK1 in FLO-1 EA cells. N = 3, ANOVA, there is no significant difference among control plasmid group, ROCK1 wild type plasmid group and ROCK1 constitutively active plasmid group.
Mentions: Next we transfected ROCK1 and ROCK2 expression plasmids into FLO-1 cells, respectively. Transfection of ROCK1 or ROCK2 plasmids remarkably increased ROCK1 or ROCK2 protein expression in FLO-1 cells, when compared with control (Fig 4A, S5 Fig and Fig 5A, S7 Fig), indicating that ROCK1 and ROCK2 proteins were successfully overexpressed in FLO-1 cells. Overexpression of the constitutively active ROCK2 significantly enhanced NOX5-S expression (Fig 4B and 4C, S6 Fig) and H2O2 production (Fig 4D) in FLO-1 EA cells, when compared with the control. However, overexpression of the constitutively active ROCK1 had no statistically significant effect on NOX5-S protein expression (Fig 5B and 5C, S8 Fig) and H2O2 production (Fig 5D) in FLO-1 EA cells, when compared with the control.

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