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.