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

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.

Fig 1. NOX5-S mRNA expression and Rho kinase activity in response to acid exposure.

(A) In organ-cultured Barrett’s mucosal biopsies, pulsed acid treatment significantly increased NOX5-S mRNA level, an increase which was significantly decreased by a Rho kinase inhibitor Y27632 (N = 3). (B) In the human esophageal adenocarcinoma cell line FLO-1, pulsed acid treatment significantly increased NOX5-S mRNA level, an increase which was significantly decreased by Y27632 (N = 3). (C) In FLO-1 EA cells (n = 6), pulsed acid treatment significantly increased Rho kinase activity in cell lysate. These data suggest that acid-induced NOX5-S expression may be mediated by activation of ROCK. ANOVA, * P<0.05, compared with pH 7.2 group; ** P<0.05, compared with pH 4 group. t test # p<0.03.

Fig 2. Role of ROCK2 in acid-induced NOX5-S expression in FLO-1 and OE33 EA cells.

(A) A typical example of three Western blot analysis and (B) summarized data showed that transfection with ROCK2 siRNA significantly decreased ROCK2 protein expression in FLO-1 cells, indicating that ROCK2 siRNA effectively knocked down ROCK2 protein expression (n = 3). (C) A typical example of three Western blot analysis and (D) summarized data showed that knockdown of ROCK2 significantly decreased acid-stimulated NOX5-S protein expression in FLO-1 cells. (E) Knockdown of ROCK2 significantly decreased acid-induced increase in NOX5-S mRNA expression in OE33 cells. (F) Knockdown of ROCK2 significantly decreased acid-induced H₂O₂ production in FLO-1 cells. The data suggest that ROCK2 kinase might mediate acid-induced NOX5-S expression and H₂O₂ production in FLO-1 EA cells. N = 3, ANOVA, * P<0.01, compared with control siRNA group; ** P<0.001 & ## p<0.05, compared with control siRNA + acid group; # P<0.05, compared with control or control siRNA group.

Fig 3. Role of ROCK1 in Acid-induced NOX5-S expression in FLO-1 EA cells.

(A) A typical example of three Western blot analysis and (B) summarized data showed that transfection with ROCK1 kinase siRNA significantly decreased ROCK1 protein expression in FLO-1 EA cells, indicating that ROCK1 siRNA effectively down-regulated ROCK1 protein expression (n = 3). (C) A typical example of three Western blot analysis and (D) summarized data showed that knockdown of ROCK1 protein did not significantly affect acid-induced NOX5-S expression. (E) Knockdown of ROCK1 protein had no statistically significant effect on acid-induced H₂O₂ production. The data suggest that ROCK1 may not be involved in acid-induced NOX5-S expression and H₂O₂ production in FLO-1 EA cells. N = 3, ANOVA, * P<0.05, compared with control siRNA group or ROCK1 siRNA group. There was no statistically significant difference between control siRNA+ acid group and ROCK1 siRNA+ acid group. # P<0.05, compared with control or control siRNA group.

Fig 4. NOX5-S expression and H₂O₂ production in FLO-1 EA cells after ROCK2 overexpression.

(A) A typical example of three Western blot analysis showed that ROCK2 protein was successfully overexpressed in FLO-1 cells after transfection with ROCK2 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of constitutively active ROCK2 significantly increased NOX5-S protein expression. (D) Overexpression of constitutively active ROCK2 significantly increased H₂O₂ production in FLO-1 EA cells. The data suggest that ROCK2 may contribute to acid-induced increase in NOX5-S expression and H₂O₂ production. N = 3, ANOVA,* P<0.03, ** P<0.001, compared with control plasmid group.

Fig 5. 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.

Fig 6. 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.

Fig 7. Role of calcium in acid-induced NOX5-S expression.

A. Acid treatment (pH 4.0, 1 hour) significantly increased NOX5-S expression in FLO-1 cells, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, B) A23187 significantly increased NOX5-S mRNA expression in FLO-1 cells. C. Acid treatment (pH 4.0, 1 hour) significantly increased NOX5-S expression in OE33 cells, an increase which was blocked by the removal of calcium with calcium free medium plus 1 mM EGTA and 1μM thapsigargin, B) A23187 significantly increased NOX5-S mRNA expression in OE33 cells. The data suggest that acid-induced NOX5-S expression may be dependent on intracellular calcium increase. N = 3, ANOVA * P<0.02 & *** P<0.01 compared with control group; ** P<0.01 & ## P<0.05, compared with acid group; paired t test # P<0.05.