Vol. 53, Issue 6, 1016-1026, June 1998

Negative Regulation of the Rat Glutathione S-Transferase A2 Gene by Glucocorticoids Involves a Canonical Glucocorticoid Consensus Sequence

K. Cameron Falkner, Thomas H. Rushmore, Mark W. Linder, and Russell A. Prough

Department of Biochemistry and Molecular Biology (K.C.F., M.W.L., R.A.P.), University of Louisville School of Medicine, Louisville, Kentucky 40292, and Merck Research Laboratory (T.H.R.), West Point, Pennsylvania 19486

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya₁ 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.