Abstract
Glucocorticoids regulate the rat glutathioneS-transferase A2 (GSTA2) gene in a biphasic manner in cultured hepatocytes that repress gene expression at low concentration (10–100 nM) but induce gene expression at high concentration (>1 μM). High concentrations of the glucocorticoid receptor (GR) antagonist RU38486 (5–10 μM) also induced the expression of GSTA2. These effects were reproduced in HepG2 cells transfected with a luciferase reporter containing 1.6 kilobase pairs of 5′-flanking sequence of GSTA2 and expression plasmids for either GR, pregnane X receptor (PXR) or a combination of both. Dexamethasone t-butylacetate (1 μM t-Bu-DEX) repressed gene expression between 60 to 75% when only GR was expressed. When PXR was expressed, both basal andt-Bu-DEX-dependent gene expression was increased over 2-fold, respectively. Biphasic regulation of gene expression was observed over a broad range of t-Bu-DEX concentrations when expression plasmids for both receptors were cotransfected. Other steroids of the pregnane class induced GSTA2 expression as expected for a PXR-dependent process. Because no canonical responsive element for the PXR-RXRα heterodimer was observed in the 5′-flanking region of the construct, deletion analysis was used to identify a pregnane responsive region between base pairs −700 and −683; this 20-bp region contains the antioxidant response element (ARE). When the ARE sequence was mutated, basal,t-butylhydroquinone- and 17α-hydroxypregnenolone-inducible expression were all lost. These results suggest that PXR interacts with factors binding to the ARE to elicit the pregnane inductive response for GSTA2.
Footnotes
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Supported by National Institutes of Health Grant DK54774 (R.A.P.) and a grant from the University of Louisville School of Medicine (K.C.F.).
- Abbreviations:
- GSTA2
- glutathione S-transferase A2
- Ah
- aryl hydrocarbon
- AhRE
- Ah-receptor response element
- ARE
- antioxidant response element
- PXR
- pregnane X receptor
- kb
- kilobase pair(s)
- DEX
- dexamethasone
- GR
- glucocorticoid receptor
- PCR
- polymerase chain reaction
- t-Bu-DEX
- dexamethasonet-butylacetate
- RU38486
- 17β-hydroxy-11β-(4-dimethylamino-phenyl)-17α-(prop-1-ynyl)-estra-4,9-dien-3-one
- DMSO
- dimethyl sulfoxide
- bp
- base pair(s)
- RXRα
- retinoid X-receptor-α
- DR3
- direct repeat separated by 3 base pairs
- QOR
- NAD(P)H:quinone oxidoreductase
- Received March 22, 2001.
- Accepted June 12, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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