Negative regulation of the rat glutathione S-transferase A2 gene by glucocorticoids involves a canonical glucocorticoid consensus sequence

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya(1) gene (gstA2) in isolated rat hepatocytes and rat liver in 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 gstA2 participate 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.