Cecil B. Pickett

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation.

ABSTRACT Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal “Neh3” domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

The Rat Quinone Reductase Antioxidant Response Element IDENTIFICATION OF THE NUCLEOTIDE SEQUENCE REQUIRED FOR BASAL AND INDUCIBLE ACTIVITY AND DETECTION OF ANTIOXIDANT RESPONSE ELEMENT-BINDING PROTEINS IN HEPATOMA AND NON-HEPATOMA CELL LINES

The antioxidant response element (ARE) found in the 5′-flanking region of the rat quinone reductase gene has been further characterized by mutational and deletion analysis. The results indicate that the 31-base pair ARE, which contains a 13-base pair palindromic sequence, can be further separated into three regions, all three of which are required for elevated basal level gene expression. These three regions include the proximal and distal half-sites as well as a 3′-flanking region consisting of 4 adenine nucleotides. Neither the proximal nor the distal half-site alone mediates transcriptional activation by β-naphthoflavone. However, when placed together the two half-sites restore responsiveness to the inducer. Interestingly, the presence of only 1 of the 4 adenine nucleotides in the 3′-flanking region of the proximal half-site is required for responsiveness to the inducer. Point mutations within the ARE indicate that several nucleotides in both the proximal and distal half-sites are required for basal level gene expression. Electrophoretic mobility shift analysis using the ARE as the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from human Hep G2 cells. Further, this complex can be detected in nuclear extracts from rat liver and rat hepatoma cells but not in mouse Hepa 1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can also be detected in F9 cells which lack significant levels of Jun/Fos proteins. Although the rat ARE resembles the human quinone reductase ARE which contains a consensus TRE, the 2-nucleotide change in the core sequence (TGACTCA versus TGACTTG) eliminates the high affinity TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in Hep G2 and other cells with different properties than AP-1.

Transcriptional regulation of rat liver glutathione S-transferase genes by phenobarbital and 3-methylcholanthrene

The relative rates of transcription of the rat liver glutathione S-transferase Ya-Yc and Yb genes were determined in purified liver nuclei isolated at different times after phenobarbital or 3-methylcholanthrene administration. The transcriptional rates of the Ya-Yc and Yb genes were elevated approximately fivefold 8 and 6 h, respectively, after phenobarbital administration. In contrast, the transcriptional rates of the Ya-Yc genes were elevated approximately eightfold at 16 h after 3-methylcholanthrene administration, whereas the transcriptional rates of the Yb genes were elevated approximately fivefold at 6 h after the administration of this xenobiotic. The elevation in transcriptional activity of the glutathione S-transferase genes is sufficient to account for the increase in glutathione S-transferase mRNA levels determined previously by RNA blot hybridization [C. B. Pickett, C. A. Telakowski-Hopkins, G. J-F. Ding, L. Argenbright, and A. Y. H. Lu (1984) J. Biol. Chem. 259, 5182-5188]. Therefore, it appears that phenobarbital and 3-methylcholanthrene elevate the level of the rat liver glutathione S-transferases primarily by augmenting the transcriptional rates of their respective genes.

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAs by 3-methylcholanthrene.

Abstract Liver poly(A + )-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.

Rat liver glutathione S-transferase B: The functional mRNAs specific for the Ya Yc subunits are induced differentially by phenobarbital

Abstract Liver poly(A + )-RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-free system in order to examine the kinetics of induction of the translatable mRNAs encoding each subunit of glutathione S -transferase B. Translatable glutathione S -transferase B mRNA levels were maximally elevated at 16 to 24 h after a single injection of phenobarbital. Interestingly, the functional mRNA specific for the low-molecular-weight subunit was elevated markedly by phenobarbital administration whereas the mRNA specific for the high-molecular-weight subunit was only increased slightly. Our data suggest that different mRNAs direct the synthesis of the two subunits of glutathione S -transferase B and that these two mRNAs are under independent regulation.

Protease activities present in wheat germ and rabbit reticulocyte lysates

Abstract Rabbit reticulocyte lysates and wheat germ lysates were found to contain significant neutral protease activity when assayed against the highly sensitive 7-amino-4-methylcoumarin (AMC) peptide substrates Phe-AMC, succinyl-Ala-Ala-Phe-AMC and t-boc-Ala-Ala-Pro-Ala-AMC (substrates for aminopeptidase, chymotrypsin and elastase-like enzymes, respectively). Additionally, wheat germ lysates contain a trypsin-like activity when assayed against CBZ-Gly-Gly-Arg-AMC and a post-proline cleaving activity which hydrolyzed the Pro-Ala bond of t-boc-Ala-Ala-Pro-Ala-AMC.

Xenobiotic responsive elements controlling inducible expression by planar aromatic compounds and phenolic antioxidants

Publisher Summary This chapter discusses the xenobiotic responsive elements (XRE) controlling inducible expression by planar aromatic compounds and phenolic antioxidants. The first xenobiotic responsive element contains the XRE core sequence found in multiple copies in the 5′ flanking region of the cytochrome P450IA1 structural gene. The second xenobiotic responsive element has no sequence similarity to the XRE core sequence. This chapter describes the methods laboratory has developed to aid in the identification and analysis of the XRE and antioxidant responsive element (ARE). It describes the procedures, reagents, and expression clones used to differentiate functionally transcriptional activation of the GST Ya subunit and quinone reductase genes through the ARE and XRE. The basic procedure used for the transfection and assay of expression vectors using calcium phosphate coprecipitation on adherent cell culture monolayers is presented. The protocol is a slightly modified version of published techniques.

Rat liver DT-diaphorase: Regulation of functional mRNA levels by 3-methylcholanthrene, trans-stilbene oxide, and phenobarbital

Total liver poly(A+)-RNA isolated from untreated, and 3-methylcholanthrene-, trans-stilbene oxide-, and phenobarbital-treated rats has been translated in the rabbit reticulocyte lysate system in order to determine the effect of these xenobiotics on the level of translationally active DT-diaphorase mRNA. The in vitro translation systems were subjected to immunoprecipitation with rabbit IgG raised against purified DT-diaphorase and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the radiolabeled, immunoprecipitated product as DT-diaphorase was confirmed by limited peptide mapping using Staphylococcus aureus V-8 protease. These quantitation results demonstrate that 3-methylcholanthrene leads to an eight-fold elevation in functional DT-diaphorase mRNA at 8 h after a single administration of 3-methylcholanthrene; whereas, trans-stilbene oxide and phenobarbital produced only a modest elevation, two- to three-fold, in the functional DT-diaphorase mRNA level. These data indicate that the increase in the level of DT-diaphorase after 3-methylcholanthrene administration noted previously [B. Höjeberg, K. Blomberg, S. Stenberg, and C. Lind (1981) Arch. Biochem. Biophys. 207, 205-216] can be totally accounted for by an elevation in the mRNA level specific for this protein.

The Antioxidant Response Element

Oxidative stress results from the production of reactive oxygen intermediates, which can arise from a number of sources including ionizing radiation, inflammation, and electrophilic xenobiotics. Quinones are highly electrophilic compounds and can be very reactive, depending on the degree of reduction. Quinones can undergo either a one-electron reduction by NADPH-cytochrome P-450 reductase to form the semiquinone free radical, a reactive oxygen intermediate, or twoelectron reduction by NAD(P)H:quinone oxidoreductase (quinone reductase, DT-diaphorase) to form the hydroquinone, a less reactive species.1 In the presence of oxygen, the semiquinone autoxidizes to form the original quinone and Superoxide anion radical. The repetition of this reaction (oxidation-reduction) can lead to oxidative stress,2 which can cause cellular damage. One cellular defense mechanism against the toxic and neoplastic effects of quinones is believed to be the enzyme quinone reductase. Rat liver cytosol from 3-methylcholanthrene (3-MC) treated rats containing high levels of quinone reductase was shown to reduce the amount of semiquinone produced in vitro using menadione as the quinone acceptor. In contrast, addition of microsomes from phenobarbital-treated rats (which contain high levels of NADPH-cytochrome P-450 reductase) or cytosol containing dicoumerol, a potent quinone reductase inhibitor, resulted in production of the semiquinone.3 Thus quinone reductase, which produces hydroquinones that can be further metabolized by conjugation and then rapidly eliminated, can function as a cellular protective mechanism against oxidative stress from xenobiotics as well as endogenous compounds.4

Isolation and characterization of a human glutathione S-transferase Ha1 subunit gene

We have isolated and characterized a human liver glutathione S-transferase Ha1 subunit gene. The gene spans approximately 12 kilobases and comprises seven exons separated by six introns. The transcription initiation site has been determined by primer extension analysis. A TATA box is located 26 nucleotides upstream from the transcription initiation site, an adenine residue. RNA blot analysis reveals that the gene is expressed at significantly higher levels in human liver than in HepG2 cells. The isolation and characterization of a human glutathione S-transferase Ha1 subunit gene should facilitate a detailed analysis of its transcriptional regulation.