Takagi Tomohisa, Naito Yuji, Okada Hitomi, Ishii Takeshi, Mizushima Katsura, Akagiri Satomi, Adachi Satoko, Handa Osamu, Kokura Satoshi, Ichikawa Hiroshi, Itoh Ken, Yamamoto Masayuki, Matsui Hirofumi, Yoshikawa Toshikazu
Kyoto Prefectural University of Medicine, Japan.
J Pharmacol Exp Ther. 2009 Oct;331(1):255-64. doi: 10.1124/jpet.109.152702. Epub 2009 Jul 23.
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.
诱导血红素加氧酶-1（HO-1）表达与质子泵抑制剂兰索拉唑的细胞保护和抗炎作用相关，但潜在的分子机制仍未完全阐明。在本研究中，我们使用培养的胃上皮细胞（大鼠胃黏膜细胞系，RGM-1），研究转录因子NF-E2相关因子2（Nrf2）及其磷酸化/激活以及 Kelch样ECH相关蛋白1（Keap1）的氧化在兰索拉唑诱导的HO-1上调中的作用。用兰索拉唑处理后，RGM-1细胞的HO-1表达以时间和剂量依赖性方式显著增强，且HO-1的这种上调有助于抑制受刺激的RGM-1细胞产生趋化因子。转染Nrf2-siRNA可抑制兰索拉唑诱导的HO-1。电泳迁移率变动分析显示，用兰索拉唑处理的RGM-1细胞中Nrf2蛋白的核转位和应激反应元件（StRE）结合活性增加。此外，在转染了含有突变StRE的HO-1增强子荧光素酶报告质粒的RGM-1细胞中，兰索拉唑诱导的HO-1报告基因活性降低。兰索拉唑促进细胞外信号调节激酶（ERK）的磷酸化，而ERK特异性抑制剂U0126可抑制兰索拉唑诱导的HO-1上调。通过Pro-Q Diamond磷蛋白富集试剂盒纯化的磷蛋白组分中检测到磷酸化的Nrf2蛋白。最后，使用生物素化半胱氨酸作为分子探针分析S-氧化蛋白，在经兰索拉唑处理的RGM-1细胞中检测到Keap1蛋白的氧化形式。这些结果表明，兰索拉唑上调大鼠胃上皮细胞中的HO-1表达，且上调的HO-1有助于该药物的抗炎作用。ERK和Nrf2的磷酸化、Nrf2的激活和核转位以及Keap1的氧化均参与兰索拉唑诱导的HO-1上调。
