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A conserved antioxidant response element (ARE) in the promoter of human carbonyl reductase 3 (CBR3) mediates induction by the master redox switch Nrf2.

ABSTRACT: Carbonyl reductase activity catalyzes the two electron reduction of several endogenous and exogenous carbonyl substrates. Recent data indicate that the expression of human carbonyl reductase 3 (CBR3) is regulated by the master redox switch Nrf2. Nrf2 binds to conserved antioxidant response elements (AREs) in the promoters of target genes. The presence of functional AREs in the CBR3 promoter has not yet been reported. In this study, experiments with reporter constructs showed that the prototypical Nrf2 activator tert-butyl hydroquinone (t-BHQ) induces CBR3 promoter activity in cultures of HepG2 (2.7-fold; p<0.05) and MCF-7 cells (22-fold; p<0.01). Computational searches identified a conserved ARE in the distal CBR3 promoter region ((-2698)ARE). Deletion of this ARE from a 4212-bp CBR3 promoter construct impacted basal promoter activity and induction of promoter activity in response to treatment with t-BHQ. Deletion of (-2698)ARE also impacted the induction of CBR3 promoter activity in cells overexpressing Nrf2. Electrophoretic mobility shift assays (EMSA) demonstrated increased binding of specific protein complexes to (-2698)ARE in nuclear extracts from t-BHQ treated cells. The presence of Nrf2 in the specific nuclear protein-(-2698)ARE complexes was evidenced in EMSA experiments with anti-Nrf2 antibodies. These data suggest that the distal (-2698)ARE mediates the induction of human CBR3 in response to prototypical activators of Nrf2.

SUBMITTER: Cheng Q

PROVIDER: S-EPMC3225504 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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A conserved antioxidant response element (ARE) in the promoter of human carbonyl reductase 3 (CBR3) mediates induction by the master redox switch Nrf2.

Cheng Qiuying Q Kalabus James L JL Zhang Jianping J Blanco Javier G JG

Biochemical pharmacology 20111005 1

Carbonyl reductase activity catalyzes the two electron reduction of several endogenous and exogenous carbonyl substrates. Recent data indicate that the expression of human carbonyl reductase 3 (CBR3) is regulated by the master redox switch Nrf2. Nrf2 binds to conserved antioxidant response elements (AREs) in the promoters of target genes. The presence of functional AREs in the CBR3 promoter has not yet been reported. In this study, experiments with reporter constructs showed that the prototypica ...[more]

PMID: 22001310

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Project description:The transcription factor NFE2L2 (Nuclear Factor, Erythroid 2-Like 2, or NRF2) plays a key role in maintaining the redox state within cells. Characterization of this pathway has extended to fish, most notably zebrafish (Danio rerio), in which two paralogs of the transcription factor exist: Nrf2a, an activator, and Nrf2b, a negative regulator during embryogenesis. Only one ARE target has been thoroughly delineated in zebrafish, and this deviated from the canonical sequence derived from studies in mammals. In general, the mechanistic pathway has not been characterized in non-model aquatic organisms that are commonly exposed to environmental pollutants. The current study compares the zebrafish paralogs to those found in a non-model teleost, the ecologically important salmonid, Oncorhnychus kisutch (coho salmon). Two salmon paralogs, Nrf2A and -2B, described here were found to possess only slightly greater identity between one another (84% of amino acids) than to the singleton ortholog of the esocid Esox lucius (80-82%), the nearest non-salmonid outgroup. Unlike one of the zebrafish forms, each is a strong activating factor based on sequence homology and in vitro testing. To uncover functional target AREs in coho, promoter flanking sequences were isolated for five genes that protect cells against oxidative stress: heme oxygenase 1, peroxiredoxin 1, glutamate-cysteine ligase, and the glutathione S-transferases pi and rho (hmox1, prdx1, gclc, gstp, and gstr). All except gstr had functional elements and all fit the standard mammalian-derived canonical sequence, unlike the motif found in zebrafish gstp. Expression studies demonstrate the presence of both Nrf2 paralogs in multiple organs, although in differing ratios. Collectively, our findings extend the conservation of Nrf2 and the ARE to salmonids, and should help inform future work in teleosts on mechanisms of redox control, as well as responsiveness of this pathway and its downstream antioxidant gene targets to chemical exposures in the environment.

Dataset Information

A conserved antioxidant response element (ARE) in the promoter of human carbonyl reductase 3 (CBR3) mediates induction by the master redox switch Nrf2.

Publications

A conserved antioxidant response element (ARE) in the promoter of human carbonyl reductase 3 (CBR3) mediates induction by the master redox switch Nrf2.

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