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 transfec tion experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acety ltransferase (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, sig nificantly 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'-f lanking region. The dexamethasone concentration dependence of the repr ession was consistent with involvement of the GC receptor and was anta gonized 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 b ut not by unrelated or mutated gstA2 GRE double-stranded oligonucleoti des. This complex was supershifted by incubation of nuclear extracts c ontaining GC receptor with anti-GC receptor globulins. Constructs cont aining multiple copies of pGRE sequence were either nonresponsive or p ositively responsive (three copies) to GC. Luciferase constructs conta ining -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated recept or 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 unidentifie d responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.