Elsevier

Methods in Enzymology

Volume 400, 2005, Pages 165-191
Methods in Enzymology

Vitamin D Receptor Regulation of the Steroid/Bile Acid Sulfotransferase SULT2A1

https://doi.org/10.1016/S0076-6879(05)00010-8Get rights and content

Abstract

SULT2A1 is a sulfo‐conjugating phase II enzyme expressed at very high levels in the liver and intestine, the two major first‐pass metabolic tissues, and in the steroidogenic adrenal tissue. SULT2A1 acts preferentially on the hydroxysteroids dehydroepiandrosterone, testosterone/dihydrotestosterone, and pregnenolone and on cholesterol‐derived amphipathic sterol bile acids. Several therapeutic drugs and other xenobiotics, which include xenoestrogens, are also sulfonated by this cytosolic steroid/bile acid sulfotransferase. Nonsteroid nuclear receptors with key roles in the metabolism and detoxification of endobiotics and xenobiotics, such as bile acid‐activated farnesoid X receptor, xenobiotic‐activated pregnane X receptor and constitutive androstane receptor, and lipid‐activated peroxisome proliferator‐activated receptor‐α, mediate transcription induction of SULT2A1 in the enterohepatic system. The ligand‐activated vitamin D receptor (VDR) is another nuclear receptor that stimulates SULT2A1 transcription, and the regulatory elements in human, mouse, and rat promoters directing this induction have been characterized. Given that bile acid sulfonation is catalyzed exclusively by SULT2A1 and that the 3α‐sulfate of the highly toxic lithocholic acid is a major excretory metabolite in humans, we speculate that a role for the VDR pathway in SULT2A1 expression may have emerged to shield first‐pass tissues from the cytotoxic effects of a bile acid overload arising from disrupted sterol homeostasis triggered by endogenous and exogenous factors.

Introduction

An elaborate network of regulated detoxification systems has evolved in humans and other mammals as a defense against the harmful overload of endobiotics and diverse xenobiotics that arise from food products to pesticides and other pollutants to clinically active drugs. The phase I metabolic products of cytochrome P450 (CYP) mixed function oxidases become more water soluble after polar groups (such as sulfate) are conjugated to the functionalized metabolites by phase II transferases. Common phase II reactions include sulfonation, glucuronidation, glutathionation, amino acylation (glycine, taurine), and alkylation (methylation, N‐acetylation). Phase II biotransformation also leads to inactivation of the reactive oxidation products of CYP‐catalyzed reactions so that damage to DNA, RNA, proteins, and other cellular entities may be averted. Indeed, induced phase I events and/or impaired phase II activities are associated with heightened risks for diseases, including cancer and neurodegenerative maladies (Bandmann 1997, Liska 1998). Cytosolically localized sulfotransferases (SULTs), glutathione S‐transferases (GSTs), N‐acetyltransferases (NATs), and membrane‐bound UDP‐glucuronosyltransferases (UGTs) are among the phase II enzymes that are well characterized regarding the genomic organization, genetic polymorphism, structural and functional diversity, and regulation by endocrine and intracrine (metabolic) factors. Phase III transporters mediating the uptake and efflux of ions and metabolites are also integral to the detoxification network; their disrupted/impaired activity is linked to the etiology of specific human diseases (Dietrich et al.., 2003).

The key roles of nuclear receptors (NRs) in the hormone‐, metabolite‐, and xenobiotic‐mediated regulation of phase I/phase II enzymes and phase III transporters have been the subject of extensive investigations in recent years. The NRs that regulate one or more of these enzymes/transporters include the bile acid‐activated receptor farnesoid X receptor (FXR), xenobiotic‐activated receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), lipid‐sensing receptor peroxisome proliferator‐activated receptor‐α ( PPAR‐α), and the anti‐inflammatory stress hormone receptor glucocorticoid receptor (GR). Vitamin D receptor (VDR)‐mediated signaling is yet another NR pathway that influences steroid and xenobiotic metabolism by inducing CYP2 and CYP3 enzymes (Adachi 2005, Drocourt 2002, Makishima 2002). The VDR‐binding regulatory sequences in promoters of the vitamin D responsive CYP2 and CYP3 genes have been characterized. The involvement of the VDR pathway extends to phase II activity, as the ligand‐activated VDR robustly induces gene transcription for SULT2A1 in liver and intestinal cells (Echchgadda et al., 2004a). SULT2A1 is a hydroxysteroid sulfotransferase with potent sulfonation activity for the steroids dehydroepiandrosterone (DHEA), testosterone, dihydrotestosterone, and pregnenolone and for amphipathic sterols bile acids, which are the catabolic end products of cholesterol metabolism.

This chapter provides a brief background on SULT2A1 and other mammalian sulfotransferases and reviews SULT2A1 regulation by several members of the NR family of transcription factors. The VDR‐mediated induction of SULT2A1 in mice and in human cell lines is described in detail, and the methods optimized in our laboratory for the investigation of SULT2A1 enzymology and gene induction and for the characterization of VDR‐responsive regulatory elements are presented.

Section snippets

Sulfotransferases and the SULT2 Family

SULT2 enzymes catalyze sulfonation of specific endogenous hydroxy‐steroids, including the prototypical substrate DHEA. The mammalian cytosolic sulfotransferases encompassing at least 47 different members are grouped as five distinct families, with further classification as subfamilies, based on primary structure homology and on preferential activity toward specific substrates (Blanchard et al., 2004). SULT enzymes catalyze the sulfate group transfer from the cofactor

SULT2A1 Enzymology

The substrate specificity of SULT2A1 was examined in our laboratory using the baculovirus‐expressed recombinant enzyme. We have developed a quick and convenient protocol for autoradiographic visualization of sulfonated substrates produced by incubation of the substrate with 35S‐labeled PAPS and the recombinant enzyme. The 35S‐labeled products are separated from free PAPS by thin‐layer chromatography (TLC). An example of the TLC autoradiogram for sulfonated steroids is shown in Fig. 1. In

Nuclear Receptors as Regulators of SULT2A1

Nuclear receptors are ligand‐inducible transcription factors with a high degree of structural and functional conservation preserved through evolution (Escriva et al., 2004). Human NRs, of which there are at least 48 members, are activated by hormones (steroids, vitamin D, retinoids, thyroid hormone) and by metabolites (cholesterol derivatives, bile acids, lipids). In addition, diverse xenobiotic molecules from pollutants to herbal products to pharmaceuticals can activate the xenobiotic‐sensing

VDR Regulation of SULT2A1

Endogenous SULT2A1 mRNA and protein expression is induced by vitamin D (Echchgadda et al., 2004a). Figure 2 shows an example of this induction in liver cells and in the mouse liver. Treatment of HepG2 hepatoma cells with vitamin D (1α,25‐dihydroxy vitamin D3) for 24 h induced SULT2A1 mRNAs ∼6‐fold in a RT‐PCR assay (Fig. 2A); in mice injected with vitamin D, Sult2A1 mRNAs in the liver increased ∼3‐fold in a Northern blot assay (Fig. 2B). This induction reflected activation of the corresponding

Regulatory Elements Directing VDR Regulation

DNase I footprinting is an informative approach for the initial mapping of the vitamin D responsive region within a long promoter fragment. Figure 6 shows the DNase I‐footprinted region from −170 to −190 positions in the mouse Sult2A1 promoter produced by recombinant VDR and RXR‐α (A: lanes 3 and 4). Incubation with BSA alone or with each receptor alone rendered no protection (A, lanes 1, 2, and 5). This region was also protected by mouse liver nuclear extracts (B, lane 2 versus lanes 1 and 3).

Conclusion

Vitamin D3 induces endogenous SULT2A1 in vivo at 10–50 nM and considering that this concentration range falls within the normal spread of the circulating vitamin D3 level in human (Feldman et al., 1997), it is highly likely that VDR also promotes basal SULT2A1 expression in enterohepatic tissues. The VDR induction of SULT2A1 may be most significant regarding the metabolic disposition of the toxic secondary bile acids, especially lithocholic acid (LCA), which is produced from chenodeoxycholic

Acknowledgments

This work was supported by NIH Grants R01‐AG–10486; R01‐AG–19660, a Merit Review grant from the Department of Veterans Affairs (VA), and a grant from the Philip Morris USA. B.C. is a Senior Career Scientist in the VA. We thank Dr. Rommel Tirona (Vanderbilt University) for the Cyp3A‐Luc construct; Dr. Ronald Evans (Salk Institute) for the RXR‐α and PXR plasmids; and Dr. David Mangelsdorf (Southwestern Medical School) for the FXR plasmid. We thank Drs. Taesung Oh and Young‐kyo Seo (for SULT2B1b

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