Regulation of the Rat UGT1A6 by Glucocorticoids Involves a Cryptic Glucocorticoid Response Element

Abstract

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 [17␤-hydroxy-11␤-(4-dimethylamino-phenyl)-17␣-(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 glucocorticoiddependent 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.The UDP-glycosyltransferases (UGTs) are a superfamily of enzymes with a molecular mass between 50 to 60 kDa (Mackenzie et al., 1997) that are located in the endoplasmic reticulum and nuclear envelope. These enzymes apparently evolved to catalyze the glucuronidation of either endogenous compounds such as bile acids, bilirubin, and steroids or xenobiotic compounds, such as metabolites of drugs and foreign chemicals (Wells et al., 2004). Many of the substrates for UGTs are oxidized metabolites formed by the cytochrome P450 system. Conjugation of the electrophilic centers of these molecules with UDP-glucuronic acids prevents protein or nucleic acid adduction and facilitates excretion by making the molecule more hydrophilic and a substrate for the anion transporter systems.UGT1A6 is of special interest because its expression can be induced by polycyclic aromatic hydrocarbons (PAHs), such as benzo-[a]pyrene, or chlorinated compounds, such as 2, 3,7,8-tetrachloro-pbenzodioxin (Emi et al., 19953,7,8-tetrachloro-pbenzodioxin (Emi et al., , 1996. These compounds constitute a major class of environmental pollutants and carcinogens and a...

Effect of Adrenalectomy on Expression and Induction of UDP-Glucuronosyltransferase 1A6 and 1A7 in Rats

“…UGT1A6 is known to be regulated by AhR and GR. 12,17) GR mRNA expression was increased by adrenalectomy (Fig. 3), which is consistent with the results of a previous report demonstrating that decreased glucocorticoid levels can be attributed to autoregulation processes such as transcriptional activation and stabilization of GR mRNA expression.…”

“…15,16) A GRE is located between base pair −141 and the promoter in the rat UGT1A6 gene. 17) A previous study using rat hepatocytes treated with dexamethasone indicated that glucocorticoids induced the expression of UGT1A6 and p-nitrophenol glucuronidation. 18) Moreover, UGT1A6 protein expression has been shown to increase after treatment with a combination of 3-MC and dexamethasone in rat hepatocytes, 18,19) indicating that glucocorticoids regulate UGT1A6 expression and/or induction.…”

Single nucleotide polymorphisms in the human corticosteroid-binding globulin promoter alter transcriptional activity

“…However, in the presence of overexpressed GRdexamethasone treatment inhibited Cbg promoter activity. Similar effects have also been reported on the promoter activity of many other genes, such as sterol 27-hydroxylase (CYP27A1) [45], CYP2C19 [46], UDPglycosyltransferase 1A6 (UGT1A6) [47] and UGT1A1 [48] in HepG2 cells. Therefore, the level of endogenous GR, which could be activated by dexamethasone and subsequently translocated into the nucleus, was insufficient for exerting a transrepression effect on human Cbg promoter, and this could be overcome by complementation with a GR expression vector.…”

Natural Products Modulate the Multifactorial Multidrug Resistance of Cancer

“…GST is the predominant enzyme found in ovarian carcinoma and several studies have been performed to determine whether levels of this enzyme have prognostic significance, where the levels of GSTs were correlated to a poor response to chemotherapy. The first linkage between PXR and GSTs observed in rat GSTA2, GSTA2 expression was suppressed at nanomolar concentration of DEX (GR activation) in cultured hepatocytes, but induced by DEX at micromolar (PXR activation) concentrations or RU486, which is a GR antagonist and PXR agonist [87] (Figure 5). More recently, studies have focused on the inhibition of apoptosis as the so-called de novo mechanism of drug resistance.…”