Molecular mechanism and functional consequences of lansoprazole-mediated heme oxygenase-1 induction

Abstract

Aim: To investigate the molecular mechanism and functional consequences of heme oxygenase-1 (HO-1) activation by lansoprazole in endothelial cells and macrophages.

Methods: Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species (ROS) formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed using in vivo bioluminescence imaging.

Results: Lansoprazole increased HO-1 mRNA levels in endothelial cells and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH-mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity.

Conclusion: Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.

Figure 1

Effect of ranitidine and lansoprazole on HO-1 mRNA induction in endothelial cells after 8 h of incubation. Fold induction from control levels is shown as mean ± SD of three separate experiments. ^aP < 0.05 vs control. Treatment with CdCl₂ was used as a positive control. Representative Northern blotting analysis is shown in the upper panel.

Figure 2

Effect of lansoprazole on HO-1 protein expression in macrophages (J774 cells) after 12 h of incubation. Fold induction from control levels is shown as mean ± SD of three to six separate experiments. ^aP < 0.05 vs control. Representative Western blotting analysis is shown in the upper panel.

Figure 3

Lansoprazole, but not ranitidine, decreased NADPH-dependent ROS formation in macrophages. This effect was reversed in the presence of CrMP. Measurements of lucigenin-enhanced chemiluminescence were performed. ^aP < 0.05 vs control; ^cP < 0.05 vs lansoprazole alone. Data are shown as mean ± SD of three to six separate experiments.

Figure 4

Lansoprazole increased ferritin protein expression in a concentration- and time-dependent manner in macrophages (A) and endothelial cells (B). Data are shown as mean ± SD of three to six separate experiments. ^aP < 0.05 vs control. A representative Western blotting analysis is shown in the upper panel.

Figure 5

Effect of SOD on lansoprazole-induced HO-1 mRNA expression. After pretreatment with SOD for 20 min, endothelial cells were incubated with lansoprazole for 8 h. Fold induction compared to control is shown as mean ± SD of three separate experiments. ^aP < 0.05 vs control. A representative Northern blotting analysis is shown in the upper panel.

Figure 6

Effect of different MAPK inhibitors on lansoprazole-induced HO-1 mRNA expression. After pretreatment with ERK inhibitor PD098059 (A), JNK inhibitor SP600125 (B), or p38 inhibitors SB203580 and SB202190 (C) for 20 min, endothelial cells were incubated with lansoprazole for 8 h. The Northern blotting analysis shown is representative of three to six independent experiments.

Figure 7

Effect of PI3K inhibitors LY294002 and wortmannin on lansoprazole-induced HO-1 mRNA expression. After pretreatment with PI3K inhibitors for 20 min, endothelial cells were incubated with lansoprazole for 8 h. Representative Northern blotting analysis is shown (A and B). Results are expressed as mean ± SD of three to six separate experiments compared to lansoprazole (control = gene expression of cells stimulated with 50 μmol/L lansoprazole = 100%) (C). ^aP < 0.05 vs lansoprazole alone.

Figure 8

Effect of PI3K-inhibitors LY294002 and wortmannin on lansoprazole-induced HO-1 promoter activity in stable transfected cell lines. Representative images of three to six separate experiments are shown (A and B). Light emission (shown by the rainbow bars: blue, least intense; red, most intense) was measured in NIH3T3-HO-1-luc cells with the IVIS. Results are expressed as mean ± SD of three to six separate experiments compared to lansoprazole (control = HO-1 promoter activity of cells stimulated with 30 μmol/L lansoprazole = 100%) (C). ^aP < 0.05 vs lansoprazole alone.