Chemical Inducers of Rodent GlutathioneS-Transferases Down-Regulate HumanGSTA1Transcription through a Mechanism Involving Variant Hepatic Nuclear Factor 1-C

Abstract

The regulation of human GSTA1 by chemical inducers of rodent glutathione S-transferases (GSTs) and the regulatory role of hepatic nuclear factor (HNF) 1 was investigated in Caco-2 cells. Treatment of preconfluent and confluent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), 3-methylcholanthrene (3-MC), 2-tert-butyl-4-hydroxy-anisol (BHA), and phenobarbital (PB) reduced GSTA1 mRNA levels in preconfluent and confluent cells. Constitutive levels of GSTA1 and HNF1␣ mRNA were elevated 6.25-and 50-fold, respectively, in postconfluent cells compared with preconfluent cells. Overexpression of HNF1␣ in cells transfected with a GSTA1 promoter-luciferase construct (pGSTA1-1591-luc) resulted in dose-related increases in reporter activity not observed when an HNF1 response element (HRE) in the proximal promoter was mutated (pGSTA1-⌬HNF1-luc). TPA, 3-MC, BHA, and PB reduced HNF1␣ mRNA levels in preconfluent and confluent cells and caused marked reductions in luciferase activity in pGSTA1-1591-luc transfectants. Transcriptional repression was abrogated with pGSTA1-⌬HNF1-luc and with truncated constructs that eliminated a functional HRE. Moreover, cotransfection of pHNF1␣ with pGSTA1-1591-luc partially prevented the reduction in luciferase activity by rodent GST inducers. Immunoblot analysis of DNA binding studies indicate that variant (v)HNF1-C binding to HRE is increased in preconfluent cells treated with 3-MC, BHA, and PB. In addition, overexpression of vHNF1-C repressed GSTA1 transcriptional activity in luciferase reporter assays. Finally, treatment with 3-MC, BHA, and PB increased vHNF1-C mRNA levels in preconfluent cells. These data demonstrate that repression of human GSTA1 transcription by chemical inducers of rodent GSTs occurs, in part, through a mechanism involving the repressive action of vHNF1-C.Glutathione S-transferases (GSTs) are a family of multifunctional proteins involved in the detoxification of a broad range of xenobiotics and therapeutic compounds, playing a critical role in protecting cells from reactive electrophiles. Rodent GSTs are inducible by drugs, carcinogens, antioxidants, and other dietary components. Inducers such as 2-tertbutyl-4-hydroxyanisole (BHA), phenobarbital (PB), 3-methylcholanthrene (3-MC), dithiolethiones, and ethoxyquin differentially control the regulation of mouse and rat hepatic GST isoenzymes and have led to the discovery of new inducible ␣-and -class subunits (Ding and Pickett, 1985;Hayes et al., 1991). Transcriptional activation of rodent ␣ class GSTs occurs via the antioxidant-responsive element (ARE), required for induction by dithiolethiones and antioxidants, as well as the xenobiotic-responsive element, which was first noted as a mediator in the induction of cytochrome P450 1A1 by polyaromatic hydrocarbons. (Rushmore et al., 1990;Hayes and Pulford, 1995;Eaton and Bammler, 1999).Only a few studies have addressed the regulation of human GST genes. Although the intron and exon structure of the two major human GSTA genes (A1 and A2) is highly homologous with tho...

Repression of HumanGSTA1by Interleukin-1β Is Mediated by Variant Hepatic Nuclear Factor-1C

“…Thus, to test this hypothesis we first verified that overexpression of vHNF-1C has a repressive effect on GSTA1 transactivation. Moreover, in a previous study we showed that vHNF-1C mRNA levels were high in preconfluent Caco-2 cells (and markedly higher than HNF-1␣) but comparatively lower in postconfluent cells (Romero et al, 2006). This finding may partially explain the differences observed in GSTA1 expression in pre-and postconfluent cells.…”

“…Experiments were repeated twice with similar results. dent GSTs occurs via a mechanism involving enhanced binding vHNF-1C to the HRE in the GSTA1 promoter (Romero et al, 2006). Taken together, these data indicate an important regulatory role for HNF-1 in GSTA1 gene regulation; however, treatment conditions may determine whether HNF-1 has repressive or transactivating function based on the differential induction and dimerization of the various isoforms of HNF-1␣ and vHNF-1.…”

“…However, evidence to date suggests that transcriptional regulation of human and rodent GSTs differs significantly because the 5Ј-flanking region of hGSTA1 and hGSTA2 genes do not contain the same regulatory elements as found in the rodent homologs. Indeed, we have shown that GSTA1 is down-regulated by chemical inducers of rodent GSTs via a mechanism involving overexpression of vHNF-1C (Romero et al, 2006) Several studies have identified a role for HNF-1 in GSTA regulation. For example, reduction of human GSTA2 levels during the acute phase response is mediated by HNF-1 (Whalen et al, 2004) and decreased GSTA2 expression in human renal cell carcinomas (Klone et al, 1990) is associated with diminished levels of HNF-1 (Clairmont et al, 1994).…”

Alterations in LMTK2, MSMB and HNF1B gene expression are associated with the development of prostate cancer

“…The HNF1B isoforms are known to exhibit differences in function and target specificities; Isoforms (A) and (B) are transcriptional activators whereas isoform HNF1B(C) is a transcriptional repressor [35]. HNF1B isoforms have also been reported to activate different targets, for example, the HNF1B(C) isoform specifically has been demonstrated to negatively regulate the Glutahione-S-transferase A ( GSTA ) promoter, via a mechanism that involves Il-1beta [36]. Given the difference in the transcriptional properties and target specificity of these isoforms, we predict that the alterations to HNF1B profile we note may manifest as an alteration to the overall activity of the HNF1B gene and/or activation of a variant set of its target genes.…”

Analysis of differentially expressed genes in malignant biliary strictures

“…Because TFs are potential targets of new anticancer drugs (Darnell Jr., 2002), we screened for the TFs that control expression of the GSTA1 gene and for their binding sites in the GSTA1 promoter. This analysis yielded identification of the 4 TFs including Sp1 (Ebert et al, 2003), AP-1 (Romero et al, 2006), c-Jun (Hayes and McMahon, 2001), and c-Fos (Tang et al, 2008) and of their binding sites in the GSTA1 promoter. The differential expression in GSTA1 genotypes defined by the C-69T substitution has been attributed to an alteration of the Sp1 transcription factor (Tijhuis et al, 2005).…”