Abstract
Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathioneS-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liverin vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between −1.6 and −1.1 kb of the 5′-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20–30%; no effect of GC was observed with CAT constructs containing −1.15 kb of the 5′-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing −1.62 to −1.03 kb of the 5′-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5′-flanking region. We conclude that pGRE and half-site GREs of gstA2participate in regulation of this gene; however, a second unidentified responsive element must exist between −1.03 and −0.164 kb, resulting in repression of gstA2 expression.
Footnotes
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Send reprint requests to: Russell A. Prough, Ph.D., Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292. E-mail:raprou01{at}ulkyvm.louisville.edu
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This work was supported in part by National Institutes of Environmental Health Sciences Grant ES04244.
- Abbreviations:
- gstA2
- glutathioneS-transferase 1 (Ya1) subunit gene
- AHRRE
- aryl hydrocarbon receptor response element
- AHR
- aryl hydrocarbon receptor
- ARE
- antioxidant response element
- BA
- 1,2-benzanthracene
- CAT
- chloramphenicol acetyltransferase
- C/EBP
- CCAAT/enhancer binding protein
- bp
- base pair(s)
- CMV
- cytomegalovirus
- DEX
- dexamethasone
- GC
- glucocorticoid
- GRE
- hexonucleotide glucocorticoid response element [TGT(T/C)CT]
- PCR
- polymerase chain reaction
- pGRE
- palindromic glucocorticoid response element
- HNF
- hepatic nuclear factor
- PAH
- polyaromatic hydrocarbon
- PPAR
- peroxisome proliferator activated receptor
- MMTV
- mouse mammary tumor virus
- Received February 4, 1998.
- Accepted February 19, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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