Human hydroxysteroid sulfotransferase or (HUMAN)SULT2A1 catalyzes the sulfonation of procarcinogen xenobiotics, hydroxysteroids, and bile acids and plays a dynamic role in hepatic cholesterol homeostasis. The treatment of primary cultured human hepatocytes with a peroxisome proliferatoractivated receptor ␣ (PPAR␣)-activating concentration of ciprofibrate (10 Ϫ4 M) increased (HUMAN)SULT2A1 mRNA, immunoreactive protein, and enzymatic activity levels by ϳ2-fold. By contrast, expression of (RAT)SULT2A3, the rat counterpart to (HUMAN)SULT2A1, was induced by treatment of primary hepatocyte cultures with an activator of the pregnane X receptor, but not PPAR␣. In HepG2 cells, transient transfection analyses of luciferase reporter constructs containing upstream regions of the (HUMAN)SULT2A1 gene implicated a candidate peroxisome proliferator response element (PPRE) at nucleotides (nt) Ϫ5949 to Ϫ5929 relative to the transcription start site. Sitedirected mutagenesis and electrophoretic mobility shift assay studies confirmed that this distal PPRE (dPPRE), a direct repeat nuclear receptor motif containing one intervening nt, represented a functional PPRE. Chromatin immunoprecipitation analysis indicated that the (HUMAN)SULT2A1 dPPRE was also a functional element in the context of the human genome. These data support a major role for the PPAR␣ transcription factor in the regulation of hepatic (HUMAN)SULT2A1. Results also indicate that important species differences govern the transactivation of SULT2A gene transcription by nuclear receptors.

The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5Ј-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] Ϫ6160 and Ϫ54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4␣ bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4␣ plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4␣ activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

ABSTRACT:The mechanism responsible for glucocorticoid receptor (GR)-mediated induction of rat hepatic hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription was investigated. We previously reported that the region of the SULT2A-40/41 5-flanking region delimited by ؊158 to ؊77 nucleotides relative to the tran-

ABSTRACT:The effects of [4-(6-allyl-methyl-amino-hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 M Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor ( A primary mode of defense that is used by animals against their chemical environments involves recognition by a "xenobiotic-sensing" receptor followed by the induction of phase I and phase II xenobiotic-metabolizing enzymes, as well as "phase III" transporters. As the archetype of this mechanism, many xenobiotics bind to the pregnane X receptor (PXR), owing to the receptor's unusually accommodating ligand-binding pocket (Watkins et al., , 2003. On ligand binding, PXR, in partnership with the retinoic X receptor, is transformed into an active transcription factor that increases the expression of target genes, which include members of the CYP3A family (e.g., CYP3A23 in rat, CYP3A11 in mouse, and CYP3A4 in human). These CYP3A enzymes catalyze the phase I metabolism of numerous xenobiotic substrates, including a large number of clinically used drugs (Quattrochi and Guzelian, 2001).In addition to serving as a xenobiotic recognition and metabolizing system, PXR and CYP3A enzymes are increasingly perceived to function in the metabolism of endogenous molecules. For example, the cholestatic secondary bile acid, lithocholate, both activates PXR and is a substrate for CYP3A (Staudinger et al., 2001;Xie et al., 2001). We have used chemical inhibitors of various steps of the cholesterol biosynthetic pathway as an approach for identifying endogenous modulators of hepatic cytochrome P450 expression (Fig. 1). In this regard, we have reported that inhibitors of squalene synthase (e.g., squalestatin 1), the first committed step in cholesterol biosynthesis, selectively induce CYP2B expression in primary cultured rat hepatocytes and rat liver through a mechanism that requires the

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The purpose of the current study was to establish the role of the glucocorticoid receptor (GR) and androgen receptor (AR) transcription factors in the transactivation of rat aryl sulfotransferase (SULT1A1) gene transcription and to identify the functional hormone-responsive element(s) in the SULT1A1 gene. A cis-acting inverted repeat with three intervening bases (IR3) was identified in the 5-flanking of the SULT1A1 gene that mediates the transactivation of SULT1A1 gene transcription by both the GR and AR. CV-1 cells were cotransfected with SULT1A1-luciferase reporter plasmids and either wild-type or mutant GR or AR expression constructs. In cotransfectants expressing the wild-type GR, treatment with triamcinolone acetonide produced an ϳ4-to 6-fold induction of luciferase activity in IR3-containing SULT1A1 reporter plasmids. IR3-containing SULT1A1 reporter constructs were also activated by treatment with the synthetic androgen R1881 in cells cotransfected with wild-type but not mutant AR. In primary cultured rat hepatocytes, androgen-inducible expression of IR3-containing SULT1A1 reporter plasmids required cotransfection with AR expression plasmid. Targeted disruption of the SULT1A1 IR3 by mutation of a conserved GT sequence in the 3 half-site of the element ablated GR and AR responsiveness. These results indicate that a proximal IR3 element in the 5-flanking region of the rat SULT1A1 gene is sufficient for the transactivation of SULT1A1 gene transcription by the GR and AR, and that relative to the GR, functional AR activity is reduced in primary cultured rat hepatocytes.