Abstract
Glucocorticoids precociously induce fetal rat UGT1A6 and potentiate polycyclic aromatic hydrocarbon (PAH)-dependent induction of this enzyme in vivo and in isolated rat hepatocytes. To establish whether induction was due to glucocorticoid receptor (GR), luciferase reporter vectors were tested in transfection assays with HepG2 cells. Using a reporter construct containing approximately 2.26 kilobases of the 5′-flanking region of the UGT1A6-noncoding leader exon (A1*), dexamethasone increased basal activity 3- to 7-fold in cells cotransfected with an expression plasmid for GR. PAH increased gene expression 23-fold, but the presence of dexamethasone only induced PAH-dependent expression by 1.5-fold, suggesting interaction between GR and the aryl hydrocarbon (Ah) receptor. Furthermore, the GR antagonist RU 38486 [17β-hydroxy-11β-(4-dimethylamino-phenyl)-17α-(prop-1-ynyl)-estra-4,9-dien-3-one] was a partial agonist that increased, rather than inhibited, basal activity 3-fold. 5′-deletion analysis defined the 5′-boundary for a functional glucocorticoid-responsive unit between base pairs –141 and –118 relative to the transcription start site. This region contains the Ah receptor response element (AhRE), and both PAH and glucocorticoid-dependent gene activation were lost when this area was deleted. Mutation of a single base pair located in the AhRE region simultaneously reduced induction by PAH and increased glucocorticoid induction. Thus, the sequences of both the AhRE and glucocorticoid response elements seem to overlap, suggesting that Ah receptor binding may decrease glucocorticoid-dependent induction due to interactions of these two cis-acting elements. Mutation of a putative GRE located between base pair –81 and –95 reduced, but did not completely eliminate, glucocorticoid-dependent induction of the reporter, suggesting that a nonclassic mechanism of induction is involved in this response.
Footnotes
-
This work was supported by National Institutes of Health Grant DK54774 (R.A.P.) and Projects 2 (R.A.P.) and 5 (K.C.F.) of National Aeronautics and Space Administration Grant NAG5-12874 (E. Wang).
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.107.018952.
-
ABBREVIATIONS: UGT, UDP-glycosyltransferase; PAH, polycyclic aromatic hydrocarbon; Ah, aryl hydrocarbon; DEX, dexamethasone; CMV, cytomegalovirus; GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus; GRE, glucocorticoid response element; PPAR, peroxisome proliferator-activated receptor; PCR, polymerase chain reaction; BA, 1,2-benzanthracene; kb, kilobase; DMSO, dimethyl sulfoxide; bp, base pair(s); LTR, long terminal repeat; AhR, aryl hydrocarbon receptor; AhRE, aryl hydrocarbon receptor response element; RU 38486, 17β-hydroxy-11β-(4-dimethylamino-phenyl)-17α-(prop-1-ynyl)-estra-4,9-dien-3-one.
- Received September 19, 2007.
- Accepted November 21, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|