Abstract
Induction of heme oxygenase-1 (HO-1) expression has been associated with cytoprotective and anti-inflammatory actions of lansoprazole, a proton pump inhibitor, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigate the role of transcriptional NF-E2-related factor 2 (Nrf2), its phosphorylation/activation, and oxidation of Kelch-like ECH-associating protein 1 (Keap1) in lansoprazole-induced HO-1 up-regulation using cultured gastric epithelial cells (rat gastric mucosal cell line, RGM-1). HO-1 expression of RGM-1 cells was markedly enhanced in a time- and dose-dependent manner by the treatment with lansoprazole, and this up-regulation of HO-1 contributed to the inhibition of chemokine production from stimulated RGM-1 cells. Transfection of Nrf2-siRNA suppressed the lansoprazole-induced HO-1. An electrophoretic mobility shift assay showed increases in the nuclear translocation and stress-response elements (StRE) binding activity of Nrf2 proteins in RGM-1 cells treated with lansoprazole. Furthermore, in RGM-1 cells transfected with HO-1 enhancer luciferase reporter plasmid containing mutant StRE, lansoprazole-induced HO-1 reporter gene activity was diminished. Lansoprazole promoted the phosphorylation of extracellular signal-regulated kinase (ERK), and lansoprazole-induced HO-1 up-regulation was suppressed by U0126, an ERK-specific inhibitor. Phosphorylated Nrf2 protein was detected in the phosphoprotein fraction purified by a Pro-Q Diamond Phosphoprotein Enrichment kit. Finally, an oxidative form of the Keap1 protein was detected in lansoprazole-treated RGM-1 cells by analyzing S-oxidized proteins using biotinylated cysteine as a molecular probe. These results indicate that lansoprazole up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 contributes to the anti-inflammatory effects of the drug. Phosphorylation of ERK and Nrf2, activation and nuclear translocation of Nrf2, and oxidation of Keap1 are all involved in the lansoprazole-induced HO-1 up-regulation.
- PPI, proton pump inhibitor
- HO-1, heme oxygenase-1
- HO, heme oxygenase
- LPS, lipopolysaccharide
- MAPK, mitogen-activated protein kinase
- Nrf2, NF-E2-related factor 2
- Keap1, Kelch-like ECH-associating protein 1
- SnPP, tin-protoporphyrin
- U0126, 1,4-diamino-2,3-dicyano-1,4-bis-(methylthio)butadiene
- ERK, extracellular signal-regulated kinase
- JNK, Jun N-terminal kinase
- SP600125, anthra[1,9-cd] pyrazol-6 (2H)-one
- IL, interleukin
- CINC-1, cytokine-induced neutrophil chemoattractant-1
- ELISA, enzyme-linked immunosorbent assay
- PBS, phosphate-buffered saline
- HRP, horseradish peroxidase
- PCR, polymerase chain reaction
- PAGE, polyacrylamide gel electrophoresis
- PVDF, polyvinylidene difluoride
- TBS-T, Tris-buffered saline and 0.1% Tween 20
- ECL, enhanced chemiluminescence
- siRNA, small interfering RNA
- EMSA, electrophoretic mobility shift assay
- StRE, stress-responsive DNA element
- bZIP, basic-leucine zipper
- SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole
- GRO, growth-regulated oncogene.
Footnotes
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This work was supported by a grant-in-aid for scientific research from the Ministry of Health, Labor and Welfare of Japan [Grant 18590694].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received March 3, 2009.
- Accepted July 22, 2009.
- © 2009 by the American Society for Pharmacology and Experimental Therapeutics
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Lansoprazole, a Proton Pump Inhibitor, Mediates Anti-Inflammatory Effect in Gastric Mucosal Cells through the Induction of Heme Oxygenase-1 via Activation of NF-E2-Related Factor 2 and Oxidation of Kelch-Like ECH-Associating Protein 1
