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.     

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xenobiotic-responsive element for the transcriptional activation of the rat Cu/Zn superoxide dismutase gene

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics.     

Involvement of the xenobiotic response element (XRE) in Ah receptor-mediated induction of human UDP-glucuronosyltransferase 1A1

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), beta-naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases -3338 to -3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at -3381/-3299. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the XRE core region (CACGCA) was deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah receptor and Arnt antibodies demonstrated that the nuclear protein was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.     

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.     

The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes

The expression and activity of the phenobarbital (PB)-inducible P-450 isozymes, P-450b and P-450e, and the major 3-methylcholanthrene (MC)-inducible form, P-450c, were studied in primary cultures of adult rat hepatocytes in T1, Leibovitz L-15 (L-15), and a modification of Waymouth 752/1 (Way) media. P-450 isozymes in initially isolated hepatocytes and control and PB-treated cultures were quantitated by Western blot analysis, and activity was determined with 7,12-dimethylbenz[a]anthracene (DMBA) as substrate. Data from the Western blot analysis correlated well with the metabolic activity toward DMBA. P-450b was consistently induced by PB in hepatocytes in T1 and to a lesser extent in Way. P-450e protein was constitutive in initially isolated cells, expressed in control cultures at a reduced level, and increased or maintained by PB in all three media. DMBA metabolite formation associated with P-450b and P-450e activity was induced by PB in hepatocytes in T1 and Way and was inhibited by antibodies to P-450b. P-450c was only infrequently expressed in freshly prepared hepatocytes, but was detected in all control and PB-treated cultures although at a much higher level in T1. Thus, the amounts of P-450 isozymes, their inducibility by PB, and their activity toward DMBA were found to be dependent on the medium. We have demonstrated enzyme induction and increased activity of the major PB-inducible isozymes in hepatocytes in T1; these are also associated with a change in the control of P-450c expression leading to enhanced constitutive expression and inducibility by phenobarbital.     

The trascriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites,the anitoxidant responsive element and xenobiotic responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces SOD1 in human liver cells. Deletion analyses showed that the promoter region between -400 and -239 was responsible for the induction, in which two different characteristic regulatory elements, the antioxidant responsive element (ARE) and xenobiotic responsive element (XRE), are located. When the cells transfected with the plasmid containing those two cis-elements, the transactivation of SOD1 promoter was about 4-fold by TCDD, whereas mutation either on the ARE or XRE elevated the promoter activity by about 2-fold. Functional analyses of these two elements by deletion, mutation in the natural context, heterologous promoter assay, and gel mobility shift assay supported the notion that the activation of the SOD1 promoter was induced by TCDD through these two regulatory elements ARE and XRE. These results alongside our previous data indicate that the induction of SOD1 in response to TCDD is mediated by either Nrf2 protein or Ah receptor protein through ARE and XRE, respectively. These results also imply that the SOD1 can be induced by dioxin either in combination with or independently of these two regulatory elements to effectively defend cells from oxidative stress.