Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes as well as anti-oxidative enzymes. In this study, we investigated the transactivation potential of different Nrf2 transactivation domain regions by using the Gal4-Nrf2 chimeras and Gal4-Luc reporter co-transfection assay system in HepG2 cells. The results indicated that chimera Gal4-Nrf2-(1–370), which contains the full transactivation domain showed very potent transactivation activity. The high transactivation activity of Gal4-Nrf2-(113–251) and the diminished transactivation activities of chimera Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) suggested that the Nrf2 N-terminal 113–251 amino acids region is critical in maintaining its transactivation activity. Overexpression of upstream MAPKs such as Raf, MEKK1, TAK1-ΔN, and ASK1 up-regulated the transactivation activities of Gal4-Nrf2-(1–370) and Gal4-Nrf2-(113–251) in a dose-dependent manner. Further investigation on the effects of the three MAPK pathways on Nrf2 transactivation domain activity demonstrated that both ERK and JNK signaling pathways stimulated the Gal4-Nrf2-(1–370) transactivation activity while the p38 pathway played a negative role. Site-directed mutagenesis studies on potential MAPK phosphorylation sites of Gal4-Nrf2-(113–251) showed no significant effect on its basal transactivation activity or the fold of induction by Raf. Interestingly, the nuclear transcription coactivator CREB-binding protein (CBP), which can bind to Nrf2 transactivation domain and can be activated by ERK cascade, showed synergistic stimulation with Raf on the transactivation activities of both the chimera Gal4-Nrf2-(1–370) and the full-length Nrf2. Taken together, this study clearly demonstrated that different segments of Nrf2 transactivation domain have different transactivation potential and different MAPKs have differential effects on Nrf2 transcriptional activity. It also suggested that the up-regulation of Nrf2 transactivation domain activity by upstream MAPKs such as Raf may not be mediated by direct phosphorylation of the Nrf2 transactivation domain, but rather by regulation of the transcriptional activity of coactivator CBP.

Many studies have implicated nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-κB1 (Nfkb1) in inflammation and cancer. However, the regulatory potential for crosstalk between these two important transcription factors in inflammation and carcinogenesis has not been explored. To delineate conserved transcription factor-binding site signatures, we performed bioinformatic analyses on the promoter regions of human and murine Nrf2 and Nfkb1. We performed multiple sequence alignment of Nrf2 and Nfkb1 genes in five mammalian species – human, chimpanzee, dog, mouse and rat – to explore conserved biological features. We constructed a canonical regulatory network for concerted modulation of Nrf2 and Nfkb1 involving several members of the mitogen-activated protein kinase (MAPK) family and present a putative model for concerted modulation of Nrf2 and Nfkb1 in inflammation/carcinogenesis. Our results reflect potential for putative crosstalk between Nrf2 and Nfkb1 modulated through the MAPK cascade that may influence inflammation-associated etiopathogenesis of cancer. Taken together, the elucidation of potential relationships between Nrf2 and Nfkb1 may help to better understand transcriptional regulation, as well as transcription factor networks, associated with the etiopathogenesis of inflammation and cancer.

Many isothiocyanates (ITCs) such as sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) are highly effectively in chemoprevention or reduction of the risk of cancer and possess antitumor activities in vitro and in vivo . The activator protein 1 (AP-1) and MAPK signaling pathways are believed to play an important role in cancer chemoprevention and chemotherapy due to their involvement in tumor cell growth, proliferation, apoptosis and survival. In the present study, we determined the effects of SFN, PEITC and AITC on AP-1 activation, and investigated the roles of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways in the regulation of AP-1 activation and cell death elicited by these ITCs in human prostate cancer PC-3 cells. SFN, PEITC and AITC each induced AP-1 activity potently and caused a significant elevation in the phosphorylation of ERK1/2, JNK1/2, Elk-1 and c-Jun. Transfection with ERK2 and upstream kinase DNEE-MEK1 activated AP-1 activity, and transfection with dominant-negative mutant ERK2 (dnERK2) potently decreased AP-1 activation induced by SFN, PEITC and AITC. Transfection with JNK1 and upstream kinase MKK7 activated AP-1 activity, and transfection with dominant-negative mutant JNK1-APF significantly attenuated AP-1 activation induced by SFN, PEITC and AITC. Pretreatment with MEK1-ERK inhibitor U0126 and JNK inhibitor SP600125 substantially attenuated the decrease in cell viability induced by SFN, PEITC and AITC. Transfection with dnERK2 and JNK1-APF significantly reversed the decrease of Bcl-2 expression elicited by these ITCs. Furthermore, transfection with dnERK2 and JNK1-APF blocked the apoptosis induced by these ITCs in PC-3 cells. Taken together, our results indicate that the activation of the ERK and JNK signaling pathways is important for transcriptional activity of AP-1 and is involved in the regulation of cell death elicited by ITCs in PC-3 cells.

Curcumin has been shown to prevent and inhibit carcinogen-induced tumorigenesis in different organs of rodent carcinogenesis models. Our objective is to study global gene expression profiles elicited by curcumin in mouse liver and small intestine as well as to identify curcumin-regulated nuclear factor E2-related factor 2 (Nrf2)–dependent genes. Wild-type C57BL/6J and Nrf2 knockout C57BL/6J/Nrf2(−/−) mice were given a single oral dose of curcumin at 1,000 mg/kg. Liver and small intestine were collected at 3 and 12 hours after treatments. Total RNA was extracted and analyzed using Affymetrix (Santa Clara, CA) mouse genome 430 array (45K) and GeneSpring 6.1 software (Silicon Genetics, Redwood City, CA). Genes that were induced or suppressed >2-fold by curcumin treatments compared with vehicle in wild-type mice but not in knockout mice were filtered using GeneSpring software and regarded as Nrf2-dependent genes. Among those well-defined genes, 822 (664 induced and 158 suppressed) and 222 (154 induced and 68 suppressed) were curcumin-regulated Nrf2-dependent genes identified in the liver and small intestine, respectively. Based on their biological functions, these genes can be classified into the category of ubiquitination and proteolysis, electron transport, detoxification, transport, apoptosis and cell cycle control, cell adhesion, kinase and phosphatase, and transcription factor. Many phase II detoxification/antioxidant enzyme genes, which are regulated by Nrf2, are among the identified genes. The identification of curcumin-regulated Nrf2-dependent genes not only provides potential novel insights into the biological effects of curcumin on global gene expression and chemoprevention but also points to the potential role of Nrf2 in these processes. [Mol Cancer Ther 2006;5(1):39–51]