Analysis of the upstream elements of the xenobiotic compound-inducible and positionally regulated glutathione S-transferase Ya gene.

In situ hybridization and other data showed that all hepatocytes express glutathione-S-transferase (GST) Ya mRNA but that specifically pericentral cells can be induced 15- to 20-fold with 3-methylcholanthrene (3-MC). In order to identify DNA sequences involved in inducible expression (pericentral hepatocytes) and constitutive expression (all hepatocytes), the upstream regions of the GST Ya gene were further analyzed by transient transfection and DNA-binding studies to identify the nature of proteins involved in regulating this gene. The sequences from -980 to -650 were necessary and sufficient for cell-specific and inducible expression. Within this enhancer region, four nuclear protein-binding sites were identified. One site required for inducible expression was bound by a protein(s) induced by 3-MC. Two other sites were bound by proteins similar or identical to the constitutive hepatocyte nuclear factors HNF1 and HNF4. The fourth site was shown to be bound by a non-liver-specific nuclear protein that is also important in the function of the albumin gene enhancer.

Regulation of microsomal, xenobiotic epoxide hydrolase messenger RNA in persistent hepatocyte nodules and hepatomas induced by chemical carcinogens.

We have utilized a DNA clone complementary to epoxide hydrolase mRNA as a probe to examine the level of the mRNA in persistent hepatocyte nodules and hepatomas induced by the Solt-Farber chemical carcinogenesis procedure. Epoxide hydrolase mRNA is increased 14-fold in nodules as compared to the level in normal liver. When rats with liver nodules were administered phenobarbital, an inducer of epoxide hydrolase mRNA in normal animals, a superinduction in epoxide hydrolase mRNA was observed in the nodules (22-fold) as compared to normal liver. Surprisingly, nodule induction in conjunction with phenobarbital administration also produced marked elevation in epoxide hydrolase mRNA levels in the tissue surrounding the nodules. Using HpaII and MspI to assess the degree of methylation of CCGG sites, we have found that the epoxide hydrolase gene is hypomethylated in nodules and hepatomas compared to the gene in normal liver tissue. Phenobarbital treatment alone increased epoxide hydrolase mRNA levels but did not result in hypomethylation of the epoxide hydrolase gene. These data further support the observation that hypomethylation of specific gene sequences occurs during chemical carcinogenesis and is correlated with an elevation in the steady state level of epoxide hydrolase mRNA in persistent hepatocyte nodules.

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.

Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants.

We have identified previously a xenobiotic-responsive element, which we termed the beta-naphthoflavone-responsive element, between nucleotide -722 and -682 in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene (Rushmore, T.H., King, R.G., Paulson, K.E., and Pickett, C.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3826-3830). The beta-naphthoflavone-responsive element is responsible for part of the transcriptional activation of the Ya subunit gene by planar aromatic compounds but has a sequence distinct from the xenobiotic-responsive element found in multiple copies in the cytochrome P-450 IA1 gene and as a single copy in the Ya subunit gene. In the present study, we demonstrate that the beta-naphthoflavone-responsive element is required for the transcriptional activation of the Ya subunit gene by phenolic antioxidants such as t-butylhydroquinone through a mechanism that does not require functional Ah receptors. Furthermore, we present evidence that planar aromatic compounds must be metabolized before they transcriptionally activate the Ya subunit gene through the beta-naphthoflavone-responsive element. The transcriptional activation of the Ya subunit gene by planar aromatic compounds requires a functional Ah receptor. These data provide evidence that transcriptional activation of the glutathione S-transferase Ya subunit gene can be mediated by a novel xenobiotic-responsive element which is directly responsive to phenolic antioxidants such as t-butylhydroquinone. Hence we have named this new xenobiotic-responsive element the antioxidant-responsive element or ARE.

Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants.

We have identified two regions in the 5'-flanking sequence of the rat quinone reductase gene that contain xenobiotic responsive elements. The DNA sequence of the first region spans nucleotides -393 to -352 of the 5'-flanking region and shares sequence identity with the xenobiotic responsive element (XRE) described for the cytochrome P-450 CYPIA1 gene. The DNA sequence of the second region spans nucleotides -434 to -404 of the 5'-flanking region of the quinone reductase structural gene. When a synthetic oligonucleotide corresponding to nucleotides -434 to -404 was inserted in front of a heterologous promoter linked to the chloramphenicol acetyltransferase structural gene, an increase in basal level expression as well as responsiveness to beta-naphthoflavone and t-butylhydroquinone, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, was observed. The sequence, -434 to -404, did not have any sequence identity with the XRE but shared a large degree of identity with the antioxidant responsive element recently described for the rat glutathione S-transferase Ya subunit gene (Rushmore, T. H., King, R. G., Paulson, K. E., and Pickett, C. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3826-3830; Rushmore, T. H., and Pickett, C. B. (1990) J. Biol. Chem. 265, 14648-14653). These results indicate that the antioxidant responsive element can be distinguished functionally from the classical XRE and is also involved in the regulation of the quinone reductase gene by planar aromatic compounds and phenolic antioxidants.

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 beta-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.

Acute and chronic hypoxia in rats. II. Effect on oxidative phosphorylation and in vitro protein synthesis in liver mitochondria and its subpopulations.

The objective of this investigation was to examine liver mitochondrial functions in rats exposed to 0.4 atm for 0, 5 and 27 days, Liver homogenates were fractionated by rate-zonal centrifugation utilizing iso-osmotic Ficoll-sucrose gradients; this eliminates loss of large and small mitochondria and makes possible the separation of mitochondria into subpopulations according to sedimentation coefficient. After pooling all mitochondrial fractions for obtaining composite determinations of the entire population, large diminutions in states 3 and 4 respiration (succinate as substrate) were obtained in day-5 and day-27 rats but no changes were evident with regard to ADP:O ratios, respiratory control indices or the capacity for in vitro protein synthesis. By examination of subpopulations of mitochondria, it was found that mitochondria are heterogeneous with regard to ADP:O ratios, respiratory control indices, states 3 and 4 respiration and the capacity for in vitro protein synthesis. The heterogeneity for each of these parameters was altered in day-5 and day-27 animals. Although states 3 and 4 respiration were depressed throughout the entire mitochondrial population for day-5 and day-27 rats, a subpopulation of mitochondria from day-27 rats showed respiratory control indices and ADP:O ratios which were higher than any subpopulation of mitochondria of either day-5 or day-0 animals.

Rat liver NAD(P)H:quinone reductase: isolation of a quinone reductase structural gene and prediction of the NH2 terminal sequence of the protein by double-stranded sequencing of exons 1 and 2.

Recently two reports [J. A. Robertson et al. (1986) J. Biol. Chem. 261, 15794-15799 and R. M. Bayney et al. (1987) J. Biol. Chem. 262, 572-575] have appeared concerning the nucleotide sequence of quinone reductase cDNA clones. Although the cDNA clones are virtually identical, they diverge in the 5' region that encodes the NH2 terminus of the protein. In order to clarify the sequence of this region, we have isolated quinone reductase clones from a rat genomic library using a cDNA clone, pDTD55, isolated and characterized by our laboratory. We have determined the sequence of exons 1 and 2 of the structural gene by double-stranded sequencing using oligonucleotide primers. The sequence of exons 1 and 2 of the quinone reductase structural gene along with our previous nucleotide sequence analysis of pDTD55 as well as conventional amino acid sequence analysis of the purified protein indicates that quinone reductase is composed of 274 amino acids with a molecular weight of 30,946. These data agree with the published sequence of lambda NMOR1 reported by Robertson et al.

Regulation of rat glutathione S-transferase Ya subunit gene expression. DNA-protein interaction at the antioxidant responsive element.

We have characterized the interaction of the antioxidant responsive element (ARE) in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene with its trans-acting factor. The ARE core sequence, 5'-ggTGACaaaGC-3', previously identified as the cis-acting element required for activation of the Ya subunit gene by planar aromatic compounds and phenolic antioxidants, is shown to be the high affinity recognition motif for a trans-acting factor(s) as determined by gel mobility shift assays as well as methylation interference and protection studies. The DNA-protein interaction appears to occur in the major groove and involves the GpG dinucleotide preceding and the G residue within the TGAC tetramer on the coding strand of the core sequence. In addition, DNase I protection analysis maps an extended region 5' from the core recognition motif, which was shown previously to be essential for basal activity of the ARE. The trans-acting factor is present in nuclear extracts from untreated and tert-butylhydroquinone-treated cells as determined by photochemical cross-linking experiments. The cross-linked protein appears to be a heterodimer with subunit molecular weights of approximately 28,000 and approximately 45,000.

Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate.

We have previously identified a novel xenobiotic responsive element, which has been termed the antioxidant responsive element (ARE), in the 5'-flanking region of the rat quinone reductase gene (Favreau, L. V., and Pickett, C. B. (1991) J. Biol. Chem. 266, 4556-4561). This element is responsible for basal level expression of the gene as well as transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds. In this communication, we demonstrate that hydrogen peroxide can act as an inducer through the ARE sequence, a phenomenon recently demonstrated for the glutathione S-transferase Ya subunit gene (Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639). To further characterize the quinone reductase ARE, we demonstrate by DNase I footprinting that in crude Hep G2 nuclear extracts a trans-acting factor exists which interacts with a region of DNA found within the 31-nucleotide ARE sequence. Furthermore, electrophoretic mobility shift assays demonstrate the presence of a specific DNA-protein complex which can be competed only by double-stranded oligonucleotides containing the ARE sequences from the quinone reductase and glutathione S-transferase Ya subunit genes. Methylation interference and protection assays indicate that several guanine residues found in the sequence GTGACTTGGC are involved in the binding of the nuclear factor(s) to the DNA. Although electrophoretic mobility shift assays indicate that the rat quinone reductase ARE does not contain a high affinity recognition site for in vitro translated c-Jun and c-Fos, 12-O-tetradecanoylphorbol 13-acetate can act as an inducer through the ARE sequence in Hep G2 cells.

The rat liver glutathione S-transferase Ya subunit gene: characterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element.

A nuclear protein from HepG2 cells (YABP) that binds the antioxidant response element (ARE), which is required for activation of the rat glutathione S-transferase (GST) Ya subunit gene by planar aromatic compounds and phenolic antioxidants, was further characterized by quantitative competition binding experiments and DNA mutational analysis. The apparent dissociation constant of the YABP-ARE complex was estimated as < or = 0.77 nM, suggesting that the YABP has very high affinity for the ARE. There is no difference in the affinity of the YABP for the ARE when HepG2 cells are treated with inducers that transcriptionally activate the GST Ya subunit gene. Quantitative competition binding analyses in conjunction with mutagenesis of the ARE revealed that an 11-nucleotide region in the 41-nucleotide ARE, 5'-GGTGACAAAGC-3', is responsible for binding to the YABP. Eight nucleotides of this core sequence are in close proximity to the YABP, indicating that there is a broader spectrum of protein contact points than those required for the transcriptional activation. van't Hoff analysis of effects of temperature on binding has revealed that the binding reaction is governed mainly by entropy changes, which could result from conformational changes in the YABP and/or the ARE upon the formation of the complex. In addition, the native molecular weight of the YABP was determined to be 74 300 using gel filtration chromatography. These data together with previous UV cross-linking data suggest that the YABP exists as a heterodimer.

Effect of phenobarbital on the level of translatable rat liver epoxide hydrolase mRNA.

Liver poly(A)+RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-fre system in order to determine the level of translationally active epoxide hydrolase (EC 3.3.2.3) mRNA. The in vitro translation systems were immunoprecipitated with rabbit IgG prepared against purified epoxide hydrolase, and the amount of epoxide hydrolase synthesized by the lysate programmed with control and phenobarbital poly(A)+RNA was quantitated. The level of translatable epoxide hydrolase mRNA is increased 3-fold after chronic phenobarbital administration. This level of induction correlates well with the 5-fold induction in catalytic activity of epoxide hydrolase (using styrene 7,8-oxide as substrate) in microsomes isolated from phenobarbital-treated rats. Therefore, we suggest that chronic phenobarbital administration increases the amount of functional epoxide hydrolase in rat liver microsomes by way of an increase in the translatable mRNA level encoding for the enzyme. We do not know whether the increase in mRNA is the result of increased transcription or messenger stability.

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit.

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.

Isolation and characterization of the human glutathione S-transferase A2 subunit gene.

We have isolated and characterized a second human liver glutathione S-transferase (GST) subunit gene. The nucleotide sequence of this gene indicates that it encodes the alpha class subunit A2, with a coding region of about 13 kb. Using reverse transcription assays it could be shown that the A2 subunit gene is expressed in human liver and HepG2 cells. The transcription initiation site has been determined by primer extension analysis. A "TATA"-sequence was found 26 nucleotides upstream from the transcription start site. A comparison of the structure of the A2 subunit gene with that of the A1 subunit gene shows significant sequence identity between the two genes. Southern blot analysis of restriction endonuclease digests of human DNA indicates that there may be several more human alpha class GST genes.

Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2.

A coordinated cellular response to oxidative stress occurs in part through transcriptional regulation via a cis-acting sequence known as the antioxidant response element (ARE). NF-E2-related factor 2 (Nrf2), a member of the Cap'n'Collar family of basic region-leucine zipper (bZIP) transcription factors, has been implicated as an essential component of an ARE-binding transcriptional complex, but the signaling pathway leading to its activation has remained unclear. Using a reporter gene assay, we found that ARE-directed transcription was activated by phorbol 12-myristate 13-acetate (PMA), but completely suppressed by staurosporine and Ro-32-0432, selective inhibitors of protein kinase C (PKC). Immunocytochemistry and subcellular fractionation revealed that PMA, like tert-butylhydroquinone (tBHQ), promoted the nuclear localization of Nrf2, a process that was blocked by staurosporine or Ro-32-0432. We showed that Nrf2, a previously unidentified kinase target, was phosphorylated in HepG2 cells. PMA transiently activated Nrf2 phosphorylation, whereas the addition of tBHQ or beta-naphthoflavone (betaNF) led to a persistent stimulation, which was abolished by staurosporine, but not by U0126 and SB203580, respective inhibitors of MEK and p38 kinases. Purified Nrf2 was phosphorylated in vitro by the catalytic subunit of PKC, or by PKC immunoprecipitated from cell lysates. Significantly, PKC precipitated from tBHQ- or betaNF-treated cells showed enhanced activity against Nrf2. These findings indicate an important role of the PKC pathway in the ARE-mediated gene expression, and suggest that PKC-directed phosphorylation of Nrf2 may be a critical event for the nuclear translocation of this transcription factor in response to oxidative stress.

Isolation and characterization of the rat glutathione S-transferase Yb1 subunit gene.

We have isolated and characterized a rat liver glutathione S-transferase Yb1 subunit gene. DNA sequence analysis of the Yb1 subunit gene indicates that it comprises eight exons separated by seven introns and spans approximately 5.0 kb. The transcription initiation site has been mapped by primer extension experiments. Transcription begins at a guanine residue 29 nucleotides downstream from a "TATA" sequence. The DNA sequences of all exons and some introns share significant sequence identity with the corresponding exons and introns in the Yb2 subunit gene characterized by Tu and co-workers [J. Biol. Chem. 263, 11389-11395 (1988)]. The isolation and characterization of the glutathione S-transferase Yb1 gene will allow for a detailed analysis of regulatory elements required for transcriptional regulation of this gene.