Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis

Abstract

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 GPE1 during hepatocarcinogenesis. Electrophoretic mobility-shift assay and footprinting analysis with wild-type 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 GPE1 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.