ABSTRACT:24-Hydroxycholesterol (24-OHChol) is a major cholesterol metabolite and the form in which cholesterol is secreted from the brain. 24-OHChol is transported by apolipoprotein E to the liver and converted into bile acids or excreted. In both brain and liver, 24-OHChol is a liver X receptor (LXR) agonist and has an important role in cholesterol homeostasis. 24-OHChol sulfation was examined to understand its role in 24-OHChol metabolism and its effect on LXR activation. 24-OHChol was conjugated by three isoforms of human cytosolic sulfotransferase (SULT). SULT2A1 and SULT1E1 sulfated both the 3-and 24-hydroxyls to form the 24-OHChol-3, 24-disulfate. SULT2B1b formed only 24-OHChol-3-sulfate.

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 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

The liver X receptor (LXR) agonists, 24(S),25-epoxycholesterol and T0901317, were previously shown to be capable of inducing CYP3A expression in primary cultured rodent hepatocytes through activation of the pregnane X receptor (PXR). In this study, the abilities of these two LXR agonists to regulate CYP3A4 and CYP2B6 mRNA expression in primary cultures of human hepatocytes were evaluated. Treatment with 10 or 30 microM of the endogenous oxysterol, 24(S),25-epoxycholesterol, had no effect on CYP3A4 mRNA content in five preparations of primary cultured human hepatocytes, while 30 microM 24(S),25-epoxycholesterol treatment increased CYP2B6 mRNA content by approximately two-fold. By comparison, treatment with the synthetic LXR agonist, T0901317, potently increased CYP3A4 and CYP2B6 mRNA levels in the human hepatocyte cultures, producing multi-fold increases at 10nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC(50) approximately 20nM, which was more than 10-fold lower than that of the potent PXR ligand, SR-12813, while treatment with 24(S),25-epoxycholesterol failed to induce reporter expression in this assay. Therefore, while 24(S),25-epoxycholesterol-mediated PXR activation and CYP3A induction does not appear to be conserved from rodent to human, T0901317 is among the most potent known activators of human PXR.

The cystolic sulfotransferse 1C3 (SULT1C3) gene was identified by computational analysis of the human genome and suggested to contain duplications of its last two exons (7a/b and 8a/b). Although the SULT1C3 isoform containing the more downstream exons 7b and 8b (SULT1C3d) has been expressed in Escherichia coli, crystallized, and characterized for activity, there is currently no evidence that SULT1C3 is expressed in any human tissue. Using reversetranscription polymerase chain reaction, we detected SULT1C3 mRNA in the colorectal adenocarcinoma cell line (LS180), colon, and small intestine, but the amplified fragment contained the more upstream exons 7a and 8a. 39-Rapid amplification of cDNA ends (RACE) confirmed that the SULT1C3 transcript expressed in LS180 cells contained exons 7a/8a, whereas 59-RACE identified a noncoding exon 1. Full-length SULT1C3 transcript containing exons 7a/8a was amplified from LS180 and intestinal RNA, and in vitro transcription-translation of the cloned cDNA indicated that translation primarily began at the first of three in-frame ATG codons. Since SULT1C3 containing exons 7a/8a (SULT1C3a) would differ by 30 amino acids from SULT1C3d containing exons 7b/8b, we considered the functional implications of expressing one or the other isoform by generating structural models based on the reported crystal structure for SULT1C3d. Comparison of the structures indicated that five of the residues forming the substrate-binding pocket differed between the two isoforms, resulting in a change in both electron density and charge distribution along the inner wall of the substrate-binding pocket. These data indicate that SULT1C3 is expressed in human intestine but suggest that the expressed isoform is likely to differ functionally from the isoform that has been previously characterized.