|Transcription factor Nrf2/MafK regulates rat placental glutathione S-transferase gene during hepatocarcinogenesis. |
Ikeda, H; Nishi, S; Sakai, M
The Biochemical journal
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.
|Thioredoxin-dependent redox regulation of the antioxidant responsive element (ARE) in electrophile response. |
Kim, YC; Yamaguchi, Y; Kondo, N; Masutani, H; Yodoi, J
Thioredoxin is a redox-regulating protein, the expression of which is induced by various forms of oxidative stress. Thioredoxin controls the interactions of various transcription factors through redox regulation. In K562 cells, we have previously reported that hemin induces activation of the thioredoxin gene by regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element (ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile stressor, activates the thioredoxin gene through the ARE. In an electrophoretic mobility shift assay, a specific Nrf2/small Maf binding complex was induced by tBHQ and bound to the ARE. Overexpression of Nrf2 increased the tBHQ-induced thioredoxin gene activation through the ARE, whereas that of Jun and Fos suppressed the activation. The tBHQ-induced ARE binding activity was completely abrogated by an oxidizing agent, diamide, whereas 2-mercaptoethanol (2-ME) reversibly recovered the inhibitory effects of diamide, suggesting that ARE binding activity is redox-dependent. Moreover, overexpression of thioredoxin enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore, ARE-mediated induction of thioredoxin expression is a mechanism of enhancing signal transduction through the ARE in electrophile-induced stress responses.
|Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation. |
Igarashi, K; Itoh, K; Hayashi, N; Nishizawa, M; Yamamoto, M
Proceedings of the National Academy of Sciences of the United States of America
Transcription factor NF-E2 activity is thought to be crucial for the transcriptional regulation of many erythroid-specific genes. The three small Maf family proteins (MafF, MafG, and MafK) that are closely related to the c-Maf protooncoprotein constitute half of the NF-E2 activity by forming heterodimers with the large tissue-restricted subunit of NF-E2 called p45. We have established and characterized murine erythroleukemia cells that conditionally overexpress MafK from a metallothionein promoter. The conditional expression of MafK caused accumulation of hemoglobin, an indication of terminal differentiation along the erythroid pathway. Concomitantly, DNA binding activities containing MafK were induced within the MafK-overexpressing cells. These results demonstrate that MafK can promote the erythroid differentiation program in erythroleukemia cells and suggest that the small Maf family proteins are key regulatory molecules for erythroid differentiation.