Cloning and characterization of human polyamine-modulated factor-1, a transcriptional cofactor that regulates the transcription of the spermidine/spermine N(1)-acetyltransferase gene. (1/1445)

The increased transcription and ultimate superinduction of the spermidine/spermine N(1)-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of approximately 1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of approximately 20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.  (+info)

Identification of NRF2, a member of the NF-E2 family of transcription factors, as a substrate for caspase-3(-like) proteases. (2/1445)

Apoptosis is mediated by members of the interleukin-1beta converting enzyme (ICE) family of proteases (caspases), which are activated by diverse stimuli, although the downstream molecular targets of caspases are still poorly understood. Using the modified yeast two-hybrid system, which we recently established to clone genes for caspase substrates, we identified NRF2 as a novel caspase substrate. NRF2 is a member of the NF-E2 family of basic region leucine-zipper transcription factors and has been shown to induce phase II detoxifying enzymes through anti-oxidant response elements. NRF2 was cleaved at two sites by recombinant caspase-3 in vitro as well as in HeLa cells during TNFalpha-mediated apoptosis. Overexpression of the C-terminal cleavage fragment containing the DNA binding and leucine-zipper domains induced apoptosis in HeLa cells. These observations suggest that NRF2 might have some role in the induction of apoptosis after cleavage by caspases.  (+info)

Nrf2 is essential for protection against acute pulmonary injury in mice. (3/1445)

Nrf2 is a member of the "cap 'n' collar" family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2(-/-) mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2(-/-) mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2(-/-) mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2(-/-) mice. The Nrf2(-/-) mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.  (+info)

Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. (4/1445)

Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) or pyrrolidine dithiocarbamate (PDTC) resulted in the up-regulation of the gamma-glutamylcysteine synthetase catalytic (GCS(h)) and regulatory (GCS(l)) subunit genes. Increased expression was associated with an increase in the binding of Nrf2 to electrophile response elements (EpRE) in the promoters of these genes. Nrf2 overexpression increased the activity of GCS(h) and GCS(l) promoter/reporter transgenes. Overexpression of an MafK dominant negative mutant decreased Nrf2 binding to GCS EpRE sequences, inhibited the inducible expression of GCS(h) and GCS(l) promoter/reporter transgenes, and reduced endogenous GCS gene induction. beta-NF and PDTC exposure also increased steady-state levels of MafG mRNA. In addition to Nrf2, small Maf and JunD proteins were detected in GCS(h)EpRE-protein complexes and, to a lesser extent, in GCS(l)EpRE-protein complexes. The Nrf2-associated expression of GCS promoter/reporter transgenes was inhibited by overexpression of MafG. Inhibition of protein synthesis by cycloheximide partially decreased inducibility by PDTC or beta-NF and resulted in significant increases in GCS mRNA at late time points, when GCS mRNA levels are normally declining. We hypothesize that, in response to beta-NF and PDTC, the GCS subunit genes are transcriptionally up-regulated by Nrf2-basic leucine zipper complexes, containing either JunD or small Maf protein, depending on the particular GCS EpRE target sequence and the inducer. Following maximal induction, down-regulation of the two genes is mediated via a protein synthesis-dependent mechanism.  (+info)

Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. (5/1445)

The antioxidant response element (ARE) mediates the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes in response to oxidative stress. Recent studies using knockout mice suggest that NF-E2-related factor 2 (Nrf2), along with small Maf proteins, binds and activates the ARE. In this study, using in vitro binding assays, Nrf2/MafK heterodimers were found to interact with high affinity to the ARE. However, distinct differences were observed when this interaction was compared with that formed with nuclear proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated ARE-mediated transcription in HepG2 cells, and this activation was further increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2 resulted in activation of the ARE, but this activation was no longer induced by tert-butylhydroquinone. Using ARE constructs with point mutations in the core sequence, we found that only mutations at the T or G nucleotides within the core (TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to dose-dependent repression of ARE activity. Activation of the ARE by Nrf2 was similarly antagonized by MafK. These data suggest that Nrf2 plays an important role mediating basal activity of the ARE but that small Maf proteins are repressors and not activators of ARE-mediated transcription.  (+info)

Antagonism between members of the CNC-bZIP family and the immediate-early protein IE2 of human cytomegalovirus. (6/1445)

The HCMV IE2 protein negatively autoregulates its own expression as well as represses the transactivation activity of p53. Using the repression domain of IE2 as bait in the yeast two-hybrid system, Nrf1 and Nrf2, members of the CNC-bZIP family, were found to be IE2-interacting proteins. Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for the interaction. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation. In transient transfection studies, transcription driven by six copies of an NF-E2 site or by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family is repressed by IE2. Importantly, the DNA binding activity of the Nrf1/MafK heterodimer is not impeded by IE2. In a parallel study, CNC-bZIP factors attenuate the negative autoregulation of IE2. The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA binding activity of IE2. Taken together, these results demonstrate the existence of antagonism between members of the CNC-bZIP family and IE2.  (+info)

Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. (7/1445)

Electrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases. Transcription factor Nrf2 was recently identified as a general regulator of one defense mechanism against such havoc. Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase, via antioxidant response elements. Using peritoneal macrophages from Nrf2-deficient mice, we show here that Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes heme oxygenase-1, A170, peroxiredoxin MSP23, and cystine membrane transport (system x(c)(-)) activity. The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells. The lack of induction of system x(c)(-) activity resulted in the minimum level of intracellular glutathione, and Nrf2-deficient cells were more sensitive to toxic electrophiles. Several stress agents induced the DNA binding activity of Nrf2 in the nucleus without increasing its mRNA level. Thus Nrf2 regulates a wide-ranging metabolic response to oxidative stress.  (+info)

Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. (8/1445)

The mouse heme oxygenase-1 (HO-1) gene, ho-1, contains two inducible enhancers, E1 and E2. Of several cell lines tested, induction of an E1/luciferase fusion construct, pE1-luc, by CdCl(2) is most pronounced in MCF-7 cells. In these cells, E1, but not E2, is necessary and sufficient for ho-1 gene activation. Exposure of MCF-7 cells to 10 micrometer CdCl(2) stimulates phosphorylation of ERK, JNK, and p38 mitogen-activated protein kinases, implicating one or more of these signaling pathways in ho-1 gene induction. SB203580, an inhibitor of p38, diminishes cadmium-stimulated pE1-luc expression and HO-1 mRNA levels by up to 70-80%. PD098059, an ERK pathway inhibitor, does not affect HO-1 mRNA induction at the highest concentration (40 micrometer) tested. Similarly, co-expression of a dominant-negative mutant of p38alpha, but not of ERK1, ERK2, JNK1, or JNK2, reduces basal and cadmium-induced pE1-luc activity. E1 contains binding sites for the activator protein-1 (Fos/Jun), Cap'n'Collar/basic leucine zipper (CNC-bZIP), and CCAAT/enhancer-binding protein (C/EBP) families of transcription factors. A dominant-negative mutant of Nrf2 (a CNC-bZIP member), but not of c-Jun or C/EBPbeta, inhibits pE1-luc activation by cadmium. Induction of the endogenous ho-1 gene is also inhibited by the Nrf2 mutant. Mutations of E1 that inhibit cadmium inducibility also suppress the trans-activation and DNA binding activities of Nrf2, and SB203580, but not PD098059, attenuates Nrf2-mediated trans-activation of pE1-luc. Taken together, these results indicate that cadmium induces ho-1 gene expression via sequential activation of the p38 kinase pathway and Nrf2.  (+info)