Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors.
Glucocorticoids regulate the rat glutathione S-transferase A2 (GSTA2) gene in a biphasic manner in cultured hepatocytes that repress gene expression at low concentration (10--100 nM) but induce gene expression at high concentration (>1 microM). High concentrations of the glucocorticoid receptor (GR) antagonist RU38486 (5--10 microM) also induced the expression of GSTA2. These effects were reproduced in HepG2 cells transfected with a luciferase reporter containing 1.6 kilobase pairs of 5'-flanking sequence of GSTA2 and expression plasmids for either GR, pregnane X receptor (PXR) or a combination of both. Dexamethasone t-butylacetate (1 microM t-Bu-DEX) repressed gene expression between 60 to 75% when only GR was expressed. When PXR was expressed, both basal and t-Bu-DEX-dependent gene expression was increased over 2-fold, respectively. Biphasic regulation of gene expression was observed over a broad range of t-Bu-DEX concentrations when expression plasmids for both receptors were cotransfected. Other steroids of the pregnane class induced GSTA2 expression as expected for a PXR-dependent process. Because no canonical responsive element for the PXR-RXR alpha heterodimer was observed in the 5'-flanking region of the construct, deletion analysis was used to identify a pregnane responsive region between base pairs -700 and -683; this 20-bp region contains the antioxidant response element (ARE). When the ARE sequence was mutated, basal, t-butylhydroquinone- and 17 alpha-hydroxypregnenolone-inducible expression were all lost. These results suggest that PXR interacts with factors binding to the ARE to elicit the pregnane inductive response for GSTA2.
Falkner
K C
KC
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Pinaire
J A
JA
Xiao
G H
GH
Geoghegan
T E
TE
Prough
R A
RA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000494:pharmacology; D017353:Gene Deletion; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D008297:Male; D015735:Mifepristone / Q000494:pharmacology; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D005938:Glucocorticoids; D006727:Hormone Antagonists; D006873:Hydroquinones; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D015735:Mifepristone; C032201:dexamethasone 21-tributylacetate; D003907:Dexamethasone; C018855:2-tert-butylhydroquinone; D005982:Glutathione Transferase