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. 1996 Dec 10;93(25):14960-5.
doi: 10.1073/pnas.93.25.14960.

Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene

R Venugopal  1 , A K Jaiswal
Affiliations

Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene

R Venugopal et al. Proc Natl Acad Sci U S A. .

Abstract

Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a "GC" box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription factors bind to the hARE. Overexpression of cDNA derived combinations of the nuclear proteins Jun and Fos or Jun and Fra1 repressed hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression in transfected human hepatoblastoma (Hep-G2) cells. Further experiments suggested that this repression was due to overexpression of c-Fos and Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) proteins in all the possible combinations were more or less ineffective in repression or upregulation of hARE-mediated gene expression. Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells significantly increased CAT gene expression from reporter plasmid hARE-thymidine kinase-CAT in transfected cells that were inducible by beta-naphthoflavone and teri-butyl hydroquinone. These results indicated that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The hARE-mediated basal expression, however, is repressed by overexpression of c-Fos and Fra1.

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Figures

Figure 1
Figure 1
Band and supershift assays. (A) Nucleotide sequences of human NQO1 gene hARE, mutant hARE and human collagenase gene TRE are shown. The mutant hARE contained mutations in the 3′ AP1 element. This mutation is known to significantly reduce the hARE-mediated CAT gene expression and β-NF induction (3). (B) The nuclear extract from Hepa-1 cells was incubated with preimmune serum and antibodies against murine c-Jun, Jun-D, c-Fos, and human Nrf1 and supershift assays performed with the 32P-end labeled hARE. Only shifted (SB) and supershifted (SSB) bands are shown.
Figure 2
Figure 2
Effect of overexpression of Jun and Fos proteins on TRE-, hARE-, and mutant hARE-mediated CAT gene expression. (A) The Hep-G2 cells were cotransfected with 10 μg of reporter plasmids TRE–tk–CAT (Left), hARE–tk–CAT (Center), and mutant hARE–tk–CAT (Right) with 10 μg of expression plasmids LNCX, LNCX–Jun and LNCX–c-Fos individually and in combinations as shown in separate experiments. The LNCX–c-Fos was also used in different concentrations (μg). (B) HeLa and F9 cells were cotransfected with 10 μg of reporter plasmid hARE–tk–CAT and different concentration of c-Fos expression plasmid LNCX–c-Fos. Five micrograms of RSV–β-gal plasmid was included in each case as internal control of transfection efficiency. Forty-eight hours after transfection, the cells were analyzed for β-gal and CAT activities.
Figure 3
Figure 3
Effect of overexpression of Jun and Fra1 nuclear proteins on hARE-mediated CAT gene expression. The Hep-G2 cells were cotransfected with reporter plasmid hARE–tk–CAT and expression plasmids LNCX–Jun, LNCX–Fra1R, and LNCX–Fra1C individually and in combinations as shown (Left). In the related experiments, the reporter plasmids hARE–tk–CAT (Center) and mutant hARE–tk–CAT (Right) were cotransfected with different concentrations of LNCX–Fra1C plasmid. The LNCX–Fra1C and not LNCX–Fra1R produces Fra1 protein upon transfection in cells. RSV–β-gal plasmid was used in each case as internal control. The transfected cells were analyzed for β-gal and CAT activities.
Figure 4
Figure 4
Effect of overexpression of nuclear protein Nrf1 on hARE-mediated CAT gene expression. (A Left and Center) The Hep-G2 cells were cotransfected with 10 μg of reporter plasmid hARE–tk–CAT (Left) and mutant hARE–tk–CAT (Center) and various concentrations (μg) of expression plasmids LNCX–Nrf1R (containing Nrf1 cDNA in reverse orientation) and Nrf1C (containing Nrf1 cDNA in correct orientation) in separate experiments as shown. (Right) The COS1 cells were cotransfected with 10 μg of reporter plasmid hARE–tk–CAT and different concentrations of expression plasmids pMT2–Nrf1R and pMT2–Nrf1C in separate experiments as shown. Five micrograms of RSV–β-gal plasmid were used in each case as control of transfection efficiency. Forty-eight hours after the transfection, the cells were analyzed for β-gal and CAT activities. (B) TLC autoradiogram of A Right.
Figure 5
Figure 5
Effect of β-NF and t-BHQ on Nrf1-mediated hARE–tk–CAT expression. The Hep-G2 and COS1 cells were cotransfected with 10 μg of reporter plasmid hARE–tk–CAT and 10 μg of expression plasmids Nrf1R (Left) or Nrf1C (Right). Five micrograms of RSV–β-gal plasmid were used in each case as control for transfection efficiency. Thirty-six hours after transfection, the cells were treated with dimethyl sulfoxide (control) or different concentrations of β-NF and t-BHQ for 12 hr and analyzed for β-gal and CAT activities.
Figure 6
Figure 6
Effect of overexpression of Nrf2 on hARE-mediated CAT gene expression and induction by β-NF and t-BHQ. (A) The Hep-G2 cells were cotransfected with 10 μg of reporter plasmid hARE–tk–CAT (Left) or mutant hARE–tk–CAT (Center) and different concentrations (μg) of expression plasmid LNCX–Nrf2R or LNCX–Nrf2C. (Right) The reporter plasmid was cotransfected with 10 μg of LNCX–Nrf2R or LNCX–Nrf2C. (B) The Hep-G2 cells were cotransfected with 10 μg of hARE–tk–CAT and 10 μg of expression plasmids Nrf1R or Nrf1C or Nrf2R or Nrf2C or Nrf1C plus Nrf2C. In each case, 5 μg of RSV–β-gal was used as transfection standard. In the right panel, the cells were treated with either dimethyl sulfoxide (control), β-NF, or t-BHQ 12 hr prior to harvesting. The transfected cells were analyzed for β-gal and CAT activities. (C) Summary of transcription factors that bind the hARE and/or effect the hARE-mediated CAT gene expression in mammalian cells. +, Positive effect; −, negative effect; and X, no effect.
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