Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis
Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis
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DOI：
10.1042/bj20031948
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发表时间：
2004-06-01
影响因子：
4.1
通讯作者：
Sakai, M
中科院分区：
文献类型：
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作者：
Ikeda, H;Nishi, S;Sakai, M
大鼠GST-P（胎盘谷胱甘肽S-转移酶）是一种II相解毒酶，在正常肝细胞中不表达，但在早期肝癌发生过程中以及肝细胞癌细胞中高度特异性诱导。以前的研究结果表明，GST-P基因活化主要由增强子元件GPE 1（GST-P增强子1）控制，但GST-P基因的特异性活化机制尚未完全了解[Morimura，Suzuki，Hochi，Yuki，Nomura，Kitagawa，Nagatsu，Imagawa和Muramatsu（1993）Proc.Natl.Natl.Acad.Sci. Acad. Sci. U.S.A.90，2065 -2068; Suzuki，Imagawa，Hirabayashi，Yuki，Hisatake，Nomura，Kitagawa和Muramatsu（1995）Cancer Res.55，2651-2655]。在本研究中，我们研究转录因子Nrf 2/MafK异源二聚体（Nrf 2代表NF-E2 p45相关因子2）作为GST-P基因的激活剂，通过GPE 1在肝癌发生过程中的作用。电泳迁移率变化分析和足迹分析与野生型GPE 1和GPE 1点突变体表明，Nrf 2/MafK异源二聚体特异性结合GPE 1报告转染实验表明，Nrf 2强烈刺激小鼠F9胚胎癌细胞和H4 IIE大鼠肝癌细胞中GST-P基因的表达。Northern-blot分析显示，随着肝癌前病变和肝癌的发展，GST-P和Nrf 2 mRNA水平逐渐升高。Keap 1（Kelch样ECH相关蛋白1），Nrf 2的抑制因子，减少了Nrf 2对GPE 1的激活，这种抑制在用亲电化合物处理后恢复。GST-P mRNA在H4 IIE细胞中的表达被亲电化合物诱导，其他II相解毒酶的mRNA的表达也是如此。染色质免疫沉淀分析表明，抗Nrf 2和抗MafK的抗体从癌前肝细胞和大鼠肝癌细胞（H4 IIE和dRLh 84）的染色质中沉淀GPEI，但不从正常肝细胞中沉淀。这些结果表明，Nrf 2/MafK异源二聚体调节GST-P基因的表达在早期肝癌发生和肝癌细胞。
The rat GST-P (placental glutathione S-transferase), a phase II detoxifying enzyme, is not expressed in normal liver cells, but is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells. Results of previous studies indicated that GST-P gene activation was mainly controlled by an enhancer element, GPE1 (GST-P enhancer 1), but the specific activation mechanism of the GST-P gene was not fully understood [Morimura, Suzuki, Hochi, Yuki, Nomura, Kitagawa, Nagatsu, Imagawa and Muramatsu (1993) Proc. Natl. Acad. Sci. U.S.A. 90,2065-2068; Suzuki, Imagawa, Hirabayashi, Yuki, Hisatake, Nomura, Kitagawa and Muramatsu (1995) Cancer Res. 55, 2651-2655]. In the present study, we investigate the transcription factor Nrf2/MafK heterodimer (where Nrf2 stands for NF-E2 p45-related factor 2) as an activator of the GST-P gene through the action of GPE 1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wildtype GPE1 and GPE1 point mutants showed that the Nrf2/MafK heterodimer specifically bound GPE1 Reporter transfection assays indicated that Nrf2 strongly stimulated GST-P gene expression in mouse F9 embryonal carcinoma cells and H4IIE rat hepatoma cells. Northern-blot analysis indicated that GST-P and Nrf2 mRNA increased in parallel with development of precancerous lesions and hepatocellular carcinoma. Keap1 (Kelch-like ECH-associated protein 1), an inhibitory factor of Nrf2, decreased the activation of GPE1 by Nrf2 and this suppression was restored after treatment with electrophilic compounds. GST-P mRNA expression in H4IIE cells was induced by electrophilic compounds, as was the expression of mRNAs of other phase II detoxifying enzymes. Chromatin immunoprecipitation analyses showed that antibodies both against Nrf2 and against MafK precipitated GPEI from the chromatin of the pre-neoplastic hepatocytes and rat hepatoma cells (H4IIE and dRLh84), but not from normal hepatocytes. These results indicate that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells.