Cis-element architecture of Nrf2-sMaf heterodimer binding sites and its relation to diseases.

Cellular detoxication is essential for health because it provides protection against various chemicals and xenobiotics. The KEAP1-NRF2 system is important for cellular defense against oxidative and electrophilic stresses as NRF2 activates the transcription of an array of cytoprotective genes, including drug-metabolizing and antioxidant enzymes, in a stress-dependent manner. The CNC family of transcription factors, including NRF2, form heterodimers with small Maf (sMaf) proteins and bind to consensus DNA sequences that have been referred to as antioxidant response element, electrophile response element, or NF-E2-binding element. These sequences are now collectively called CNC-sMaf binding element (CsMBE). In addition to forming a heterodimer with CNC proteins, sMaf proteins can form homodimers and recognize regulatory motifs called Maf recognition element (MARE). Although the CsMBE sequence substantially overlaps with that of MARE, the sequences differ. NRF2 selectively recognizes CsMBE, which is critical for cytoprotection. Recent advances in high-throughput sequencing and population-scale genome analysis provide new insights into the transcriptional regulation involved in the stress response. The integration of a genome-wide map of NRF2 occupancy with disease-susceptibility loci reveals the associations between polymorphisms in CsMBE and disease risk, information useful for the personalized medicine of the future.

Microcystin induction small Maf protein involve in transcriptional regulation of GST from freshwater mussel Cristaria plicata.

The small Mafs, MafF, MafG and MafK play critical roles in morphogenesis and homeostasis through associating with Cap "n" Collar family of transcription factors. In this study, we tried to identify a small Maf protein in the freshwater mussel Cristaria plicata. The MafK cDNA of C. plicata, designated as CpMafK, was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR. The full length cDNA of CpMafK is 2170 bp, which includes an open reading frame of 570 bp, encoding 189 amino acids. CpMafK possesses four conserved domains and shows a low level (54-63%) of sequence similarity to small Mafs from other species. The results of Real-time quantitative PCR revealed that CpMafK mRNA was constitutively expressed in tissues, and the highest expression level was in hepatopancreas. After microcystin challenge, the expression levels of CpMafK mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMafK was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). CpMafK could combine to the promoters of CpGST1 and CpGST2 with high-affinity in vitro. Therefore, CpMafK could regulate the expression of detoxification.

Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells.

AIMS/HYPOTHESIS: Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress. METHODS: MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1(-/-), Id3(-/-) and diabetic db/db mice were used. RESULTS: ID1-4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition. CONCLUSIONS/INTERPRETATION: Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant-mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2-small MAF pathway.

Declining signal dependence of Nrf2-MafS-regulated gene expression correlates with aging phenotypes.

Aging is a degenerative process characterized by declining molecular, cell and organ functions, and accompanied by the progressive accumulation of oxidatively damaged macromolecules. This increased oxidative damage may be causally related to an age-associated dysfunction of defense mechanisms, which effectively protect young individuals from oxidative insults. Consistently, older organisms are more sensitive to acute oxidative stress exposures than young ones. In studies on the Drosophila Nrf2 transcription factor CncC, we have investigated possible causes for this loss of stress resistance and its connection to the aging process. Nrf2 is a master regulator of antioxidant and stress defense gene expression with established functions in the control of longevity. Here, we show that the expression of protective Nrf2/CncC target genes in unstressed conditions does not generally decrease in older flies. However, aging flies progressively lose the ability to activate Nrf2 targets in response to acute stress exposure. We propose that the resulting inability to dynamically adjust the expression of Nrf2 target genes to the organism's internal and external conditions contributes to age-related loss of homeostasis and fitness. In support of this hypothesis, we find the Drosophila small Maf protein, MafS, an Nrf2 dimerization partner, to be critical to maintain responsiveness of the Nrf2 system: overexpression of MafS in older flies preserves Nrf2/CncC signaling competence and antagonizes age-associated functional decline. The maintenance of acute stress resistance, motor function, and heart performance in aging flies overexpressing MafS supports a critical role for signal responsiveness of Nrf2 function in promoting youthful phenotypes.

The small MAF transcription factors MAFF, MAFG and MAFK: current knowledge and perspectives.

The small MAFs, MAFF, MAFG and MAFK have emerged as crucial regulators of mammalian gene expression. Previous studies have linked small MAF function, by virtue of their heterodimerization with the Cap 'n' Collar (CNC) family of transcription factors, to the stress response and detoxification pathways. Recent analyses have revealed a complex regulatory network involving small MAF transcription factors and other cellular proteins. The expression and activity of small MAFs are tightly regulated at multiple levels. With regard to their clinical importance, small MAFs have been linked to various diseases, such as diabetes, neuronal disorders, thrombocytopenia and carcinogenesis. A better understanding of the molecular mechanisms governing the activity of small MAFs will provide novel insights into the control of mammalian transcription and may lead to the development of novel therapeutic strategies to treat common human disorders.

The Keap1-Nrf2 system as an in vivo sensor for electrophiles.

The Keap1-Nrf2 regulatory system plays a central role in cytoprotection from electrophilic and oxidative stress. In unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3-Keap1 ubiquitin E3 ligase complex and is degraded in the proteasome. Upon the exposure to electrophilic and oxidative stress, reactive cysteine residues in Keap1 are covalently modified, which abrogates the E3 ligase activity of the Cul3-Keap1 complex. Consequently Nrf2 is stabilized and induces the transcription of various cytoprotective genes. Structural analyses have revealed the overall structure of the Keap1 homodimer as well as structural features of the association between Keap1 and Nrf2, which has greatly enhanced our understanding of the molecular mechanisms involved in the regulation of the Keap1-Nrf2 system. Recently nitric oxide signaling has been shown to activate Nrf2, suggesting that Nrf2 is a mediator of the cytoprotective effect of nitric oxide. Analyses of Nrf2-null mice have revealed a critical contribution of Nrf2 to the protection from various diseases caused by electrophilic and oxidative stress. In contrast, constitutive activation of Nrf2 has been found in many cancers, resulting in resistance against chemotherapy and radiotherapy in cancer cells. Thus, Nrf2 is a promising target for drug development. The development of Nrf2 inducers and inhibitors is an important challenge for enhancing therapies for stress-induced diseases and cancers, respectively.

Molecular determinants for small Maf protein control of platelet production.

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

Discovery of the negative regulator of Nrf2, Keap1: a historical overview.

An antioxidant response element (ARE) or an electrophile responsive element (EpRE) regulate the transcriptional induction of a battery of drug-detoxifying enzymes that are protective against electrophiles. Based on the high similarity of the ARE consensus sequence to an erythroid gene regulatory element NF-E2 binding site, we have found that the transcription factor Nrf2 is indispensable for the ARE-mediated induction of drug-metabolizing enzymes. Recent genome-wide analysis demonstrated that Nrf2 regulates hundreds of genes that are involved in the cytoprotective response against oxidative stress. In-depth analysis of Nrf2 regulatory mechanisms has led us to the discovery of a novel protein, which we have named Keap1. Keap1 suppresses Nrf2 activity by specifically binding to its evolutionarily conserved N-terminal Neh2 regulatory domain. In this review article, we summarize the findings and observations that have lead to the discovery of the Nrf2-Keap1 system. Furthermore, we briefly discuss the function of the Nrf2-Keap1 system under the regulation of the endogenous electrophilic compound 15-deoxy-Δ¹²(,)¹4-prostaglandin J2. We propose that Nrf2-Keap1 plays a significant physiological role in the response to endogenous, environmental, and pharmacological electrophiles.

Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif.

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP-ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that under the physiological condition, the NESzip motif can be switched-off by heterodimerization.

Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro.

We tested the hypothesis that stress responses mediated by the Nrf2-antioxidant responsive element (ARE) pathway are involved in the initiation of retinal neuroprotection provided by bright-cyclic-light rearing. Albino rats born and raised in dim (5 lux) or bright (400 lux) cyclic light were exposed to damaging light (3000 lux, 6 h). After exposure, the outer nuclear layer thickness and area and the electroretinogram a- and b-wave amplitudes were significantly reduced in the dim-light-reared rats compared to the bright-light-reared rats, demonstrating a light adaptation neuroprotection phenomenon. In bright-cyclic-light-reared rats, the retinal levels of thioredoxin (Trx) (2.4-fold), Trx reductase (TrxR) (2.9-fold), and proteins modified by 4-hydroxynonenal (4-HNE) (1.5-fold) were upregulated by Western blot analyses, and the nuclear translocation of Nrf2 (2.2-fold) and the DNA binding activity of Nrf2, small Maf, and cJun to the ARE were increased as determined by electrophoretic mobility shift assays. In mouse photoreceptor-derived 661W cells, pretreatment with a sublethal dose of 4-HNE protected against H(2)O(2)-induced cell damage. Treatment with 4-HNE upregulated cellular Trx, TrxR, and heme oxygenase-1 (HO-1) levels in addition to DNA binding activity of Nrf2, small Maf, and cJun to the ARE. Downregulation of Nrf2 using RNA interference technology diminished 4-HNE-mediated upregulation of Trx and Trx reductase but did not affect the upregulation of HO-1 by 4-HNE. Cytoprotection by 4-HNE pretreatment against H(2)O(2)-induced cell damage was not observed in 661W cells with a silenced Nrf2 gene. The results suggest that upregulation of the Trx system by 4-HNE via the Nrf2-ARE pathway may be involved in the molecular mechanism of the retinal neuroprotection phenomenon.

Regulation of the MAFF transcription factor by proinflammatory cytokines in myometrial cells.

The MAF (proto-)oncogene family of basic-leucine zipper transcription factors plays crucial roles in the control of mammalian gene expression and development. Here we analyzed the regulation of the human MAFF gene, coding for a small MAF transcription factor, in uterine smooth muscle cells. We found that MAFF transcript levels are induced by proinflammatory cytokines in PHM1-31 myometrial cells. We observed an important induction by interleukin 1 beta (IL1B) and a weaker upregulation by tumor necrosis factor (TNF), whereas interleukin 6 (IL6) treatment had no effect. Time course experiments revealed a rapid induction of MAFF transcripts within 30 min following IL1B treatment. The presence of actinomycin D inhibited the upregulation, suggesting that regulation of MAFF mRNA levels occurs at the transcriptional level. We generated a MAFF-specific antiserum and determined that MAFF protein was also induced by TNF and IL1B in PHM1-31 cells. In contrast, it was particularly interesting that the transcript and protein levels of the highly homologous MAFG and MAFK genes are not modulated by these cytokines. Our results suggest a possible specific role for MAFF in proinflammatory cytokine-mediated control of myometrial gene expression and provide the first link between a small MAF transcription factor and the inflammatory response.