1: FASEB J. 2003 Mar;17(3):473-5. Epub 2003 Jan 2. Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression. Dickinson DA, Iles KE, Zhang H, Blank V, Forman HJ. Department of Environmental Health Sciences, School of Public Health, and Center for Free Radical Biology, University of Alabama at Birmingham, 35294-0022, USA. Dietary use of curcumin, the active component of tumeric, one of the most widely used spices, is linked to several beneficial health effects, although the underlying molecular mechanisms remain largely unknown. Correlations have been established between curcumin exposure and increases in enzymes for glutathione synthesis, particularly glutamate-cysteine ligase (GCL), and metabolism as well as glutathione content, suggesting the eliciting of an adaptive response to stress. In this study, using HBE1 cells, we found that the mechanism of curcumin-induced GCL elevation occurred via transcription of the two Gcl genes. Gcl transcription has been shown in several systems to be mediated through binding of transcription factor complexes to TRE and EpRE elements. Studies herein showed that curcumin caused modest but sustained increases in binding of proteins to DNA sequences for both cis elements but, more importantly, altered the compositions and nuclear content of proteins in these complexes. Curcumin exposure increased JunD and c-Jun content in AP-1 complexes and increased JunD while decreasing MafG/MafK in EpRE complexes. Thus, the beneficial effects elicited by curcumin appear to be due to changes in the pool of transcription factors that compose EpRE and AP-1 complexes, affecting gene expression of GCL and other phase II enzymes. PMID: 12514113 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Am J Physiol Renal Physiol. 2003 Apr;284(4):F743-52. Epub 2002 Nov 26. Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2. Alam J, Killeen E, Gong P, Naquin R, Hu B, Stewart D, Ingelfinger JR, Nath KA. Department of Molecular Genetics, Ochsner Clinic Foundation, New Orleans 70121, USA. jalam@ochsner.org The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene. PMID: 12453873 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Arch Biochem Biophys. 2002 Sep 15;405(2):265-74. Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite. Gong P, Stewart D, Hu B, Vinson C, Alam J. Department of Molecular Genetics, Ochsner Clinic Foundation, 1516 Jefferson Highway, New Orleans, LA 70121, USA. The mechanism of heme oxygenase-1 (ho-1) gene activation by arsenite was examined. Arsenite-stimulated expression of a ho-1 promoter/luciferase chimera in a dose-dependent manner in mouse hepatoma (Hepa) cells. Mutation analyses identified the arsenite-responsive sequence as the stress-response element (StRE), which resembles the binding sites for the AP-1 superfamily of basic-leucine zipper factors. In electrophoretic mobility shift assays, up to seven specific StRE-protein complexes were routinely detected using extracts from untreated Hepa cells whereas a single complex was typically observed after treatment with arsenite. Antibody "supershift" experiments identified Nrf2, JunD, and ATF3 in control complexes and the amount of these factors increased significantly in the arsenite-induced complex. MafG, ATF2, FosB, and JunB were also detected in the arsenite complex. Activation of a StRE-dependent luciferase gene by arsenite was inhibited to varying degrees by dominant-negative mutants of Nrf2, MafK, c-Fos, and CREB but most strongly with the latter. Together, these results implicate multiple basic-leucine zipper transcription factors in ho-1 gene activation by arsenite. PMID: 12220541 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Biol Chem. 2001 Jul 20;276(29):27018-25. Epub 2001 May 16. Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent mechanism in Chinese hamster ovary cells: regulation by Nrf2 and MafG transcription factors. Gong P, Hu B, Stewart D, Ellerbe M, Figueroa YG, Blank V, Beckman BS, Alam J. Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121, USA. We have shown previously that activation of the heme oxygenase-1 (ho-1) gene by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking HIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia-mimetic CoCl(2) was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIF-independent mechanisms for ho-1 gene activation by hypoxia and CoCl(2). In Ka13 cells, CoCl(2) stimulated expression of a luciferase reporter gene under the control of a 15-kilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified the cobalt-responsive sequences as the stress-response elements (StREs). In electrophoretic mobility shift assays, two specific StRE-protein complexes were observed using extracts from Ka13 cells. In response to cobalt, the level of the slower migrating complex X increased, whereas that of complex Y decreased, in a time-dependent manner. Members of the AP-1 superfamily of basic-leucine zipper factors bind to the StRE. Antibody supershift electrophoretic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of complex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but not of other bZIP factors, attenuated cobalt-mediated gene activation. Additional experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 but is dependent on cellular oxidative stress. Unlike cobalt, hypoxia did not stimulate pHO15luc expression and did not increase StRE binding activity, indicating distinct mechanisms for ho-1 gene activation by cobalt and hypoxia in Chinese hamster ovary cells. PMID: 11356853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Biol Chem. 2001 Jan 5;276(1):819-26. A set of Hox proteins interact with the Maf oncoprotein to inhibit its DNA binding, transactivation, and transforming activities. Kataoka K, Yoshitomo-Nakagawa K, Shioda S, Nishizawa M. Department of Virology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku 108-8639, Tokyo, Japan. kkataoka@bio.titech.ac.jp Maf oncoprotein is a basic-leucine zipper (bZip) type of transcriptional activator. Since many transcription factors are known to form functional complexes, we searched for proteins that interact with the DNA-binding domain of Maf using the phage display method and identified two homeodomain-containing proteins, Hoxd12 and MHox/Prx1/Phox1/Pmx1. Studies with mutants of Hox and Maf proteins showed that they associate through their DNA-binding domains; the homeodomain of Hox and the bZip domain of Maf, respectively. Reflecting the high similarity of the bZip domain, all other Maf family members tested (c-/v-Maf, MafB, MafK, MafF, and MafG) also associated with the Hox proteins. Pax6, whose homeodomain is relatively similar to MHox, also could interact with Maf. However, two other bZip oncoproteins, Fos and Jun, failed to associate with the Hox proteins, while a distantly related Hox family member, Meis1, could not interact with Maf. Through interactions with the bZip domain, the Hox proteins inhibited the DNA binding activity of Maf, whereas the binding of Hox proteins to their recognition sequences was not abrogated by Maf. We further showed that coexpression of the Hox proteins repressed transcriptional activation and transforming activity of Maf. These results suggested that the interaction of a set of Hox proteins with Maf family members may interfere not only with their oncogenicity but also with their physiological roles. PMID: 11036080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Oncogene. 1996 Jan 4;12(1):53-62. Transactivation activity of Maf nuclear oncoprotein is modulated by Jun, Fos and small Maf proteins. Kataoka K, Noda M, Nishizawa M. Department of Viral Oncology, Cancer Institute, Tokyo, Japan. The v-maf oncogene encodes a nuclear bZip protein which specifically recognizes relatively long palindromic sequences related to an AP-1 site. In this study, we investigated the relationship of transactivation and transformation activity of Maf. The amino-terminal two thirds of the molecule were dispensable for its DNA-binding activity but conferred its transactivation potential. Transactivation activities of a set of deletion mutants correlated well with their cell transforming abilities. However, a point mutant associated with enhanced oncogenic activity was not more effective in transactivation than the wild type, suggesting that some other function(s) of Maf is also important for its transforming ability. We also examined the effect of other bZip proteins on the transactivation activity of Maf. Three small Maf family proteins (MafK, MafF and MafG), which are missing the transactivation domain of v-Maf, competitively inhibited transactivation by Maf. Co-expression of Jun or Fos also affected the transactivation potential of Maf by forming Maf/Jun or Maf/Fos heterodimers of distinct DNA-binding specificities. In addition to these factors, we noticed the presence of a strong endogenous transactivating activity associated with a sequence related to an NF-E2 site rather than the typical AP-1 site in fibroblast cells. These results indicate that AP-1 site-like cis-regulatory elements of eukaryotic genes are regulated by multiple sets of bZip dimers with different DNA-binding and transactivation properties. PMID: 8552399 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Mol Cell Biol. 1995 Apr;15(4):2180-90. Erratum in: Mol Cell Biol 1995 Jun;15(6):3461. Small Maf proteins heterodimerize with Fos and may act as competitive repressors of the NF-E2 transcription factor. Kataoka K, Igarashi K, Itoh K, Fujiwara KT, Noda M, Yamamoto M, Nishizawa M. Department of Viral Oncology, Cancer Institute, Tokyo, Japan. The maf oncogene encodes a bZip nuclear protein which recognizes sequences related to an AP-1 site either as a homodimer or as heterodimers with Fos and Jun. We describe here a novel maf-related gene, mafG, which shows extensive homology with two other maf-related genes, mafK and mafF. These three maf-related genes encode small basic-leucine zipper proteins lacking the trans-activator domain of v-Maf. Bacterially expressed small Maf proteins bind to DNA as homodimers with a sequence recognition profile that is virtually identical to that of v-Maf. As we have previously described, the three small Maf proteins also dimerize with the large subunit of NF-E2 (p45) to form an erythroid cell-specific transcription factor, NF-E2, which has distinct DNA-binding specificity. This study shows that the small Maf proteins can also dimerize among themselves and with Fos and a newly identified p45-related molecule (Ech) but not with v-Maf or Jun. Although the small Maf proteins preferentially recognize the consensus NF-E2 sequence as heterodimers with either NF-E2 p45, Ech, or Fos, these heterodimers seemed to be different in their transactivation potentials. Coexpression of Fos and small Mafs could not activate a promoter with tandem repeats of the NF-E2 site. These results raise the possibility that tissue-specific gene expression and differentiation of erythroid cells are regulated by competition among Fos, NF-E2 p45, and Ech for small Maf proteins and for binding sites. PMID: 7891713 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------