H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1.

The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry.

Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells.

Pyrrolidine dithiocarbamate (PDTC) induction of the human glutamate cysteine ligase modulatory (GCLM) gene is dependent on activation of the mitogen-activated protein kinases (MAPKs) extracellular regulated kinase (Erk) and p38, and is not affected by protein kinase C (PKC) or PI3K inhibitors. Nrf2 binding to the electrophile response element (EpRE) located within the GCLM promoter is decreased after MAPK inhibition, suggesting that Nrf2 could be a downstream target of activated MAPK. To evaluate this hypothesis, a series of Nrf2 proteins harboring mutations in conserved consensus MAPK phosphorylation sites were developed and used in multiple functional assays. All mutated Nrf2 proteins tested interacted with the cytoplasmic repressor Keap1 in a manner indistinguishable from wild-type Nrf2. Furthermore, the mutant and wild-type Nrf2 proteins were similarly capable of transactivating an EpRE-containing GCLM/luciferase reporter transgene. Collectively these functional assays suggest that Nrf2 is not likely to be a direct downstream target of activated MAPK in vivo. However, treatment of HepG2 cells with MAPK inhibitors PD98059 and/or SB202190 prior to exposure to PDTC, reduced Nrf2 translocation to the nucleus, suggesting that MAPK-directed phosphorylation is a requirement for nuclear localization during PDTC induction of GCLM gene expression.

The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm.

Transactivation of phase II detoxification enzymes and antioxidant proteins is mediated by the Cap'N'Collar transcription factor, Nrf2, which is sequestered in the cytoplasm by the actin-binding protein Keap1. Mutation of a conserved serine (S104A) within the Keap1 BTB/POZ domain disrupts Keap1 dimerization and eliminates the ability of Keap1 to sequester Nrf2 in the cytoplasm and repress Nrf2 transactivation. Disruption of endogenous Keap1 dimerization using BTB/POZ dominant negative proteins also inhibits the ability of Keap1 to retain Nrf2 in the cytoplasm. Exposure to an electrophilic agent that induces Nrf2 release and nuclear translocation disrupts formation of a Keap1 complex in vivo. Collectively, these data support the conclusion that Keap1 dimerization is required for Nrf2 sequestration and transcriptional repression. Furthermore, exposure to inducing agents disrupts the Keap1 dimerization function and results in Nrf2 release.

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.

Differential induction of mafF, mafG and mafK expression by electrophile-response-element activators.

The three small Maf proteins, MafF, MafG and MafK, have been implicated in a number of physiological processes, including development, differentiation, haematopoiesis and stress response. Here we report the constitutive expression of mafF, mafG and mafK in six human cell lines derived from various tissues (HepG2, IMR-32, K-562, HEK-293, RD and A549). The expression patterns of mafF, mafG and mafK varied widely among cell lines. Because small Maf proteins have been implicated in electrophile response element (EpRE)-mediated stress response, the ability of three EpRE activators [pyrrolidinedithiocarbamate (PDTC), phenylethyl isothiocyanate (PEITC) and t-butylhydroquinone (tBHQ)] to induce small Maf expression was examined in detail in HepG2 cells. Both PDTC and PEITC induced mafF, mafG and mafK expression, whereas tBHQ failed to markedly induce any of the three small Mafs. Where a response was observed, mafF was induced to the greatest extent compared with mafG and mafK, and this response was transcriptionally mediated. PDTC also induced small Maf expression in the other cell lines examined, with patterns of induction varying among cell lines. The differences in expression among the cell lines examined, coupled with the induction patterns observed, indicate that the three small maf genes are stress-responsive, but may be regulated via differing mechanisms. Furthermore, the fact that tBHQ, PDTC and PEITC induce EpRE activity, but that tBHQ fails to markedly induce any of the small Mafs, suggests that up-regulation of small Mafs is not an absolute requirement for EpRE-mediated gene expression.