Aryl-(SULT1A1), estrogen-(SULT1E1), and hydroxysteroid-(SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre-and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre-and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre-and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

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.

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.

Objective 25-hydroxycholesterol (25HC) and its sulfated metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), regulate certain aspects of lipid metabolism in opposite ways. Hence, the enzyme for the biosynthesis of 25HC3S, oxysterol sulfotransferase (SULT2B1b), may play a crucial role in regulating lipid metabolism. We evaluate the effect of 25HC sulfation on lipid metabolism by overexpressing the gene encoding SULT2B1b in human aortic endothelial cells (HAECs) in culture. Methods and Results The human SULT2B1b gene was successfully overexpressed in HAECs following infection using a recombinant adenovirus. HPLC analysis demonstrated that more than 50% of 3H-25HC was sulfated in 24 hrs following overexpression of the SULT2B1b gene. In the presence of 25HC, SULT2B1b overexpression significantly decreased mRNA and protein levels of LXR, ABCA1, SREBP-1c, ACC-1, and FAS, which are key regulators of lipid biosynthesis and transport; and subsequently reduced cellular lipid levels. Overexpression of the gene encoding SULT2B1b gave similar results as adding exogenous 25HC3S. However, in the absence of 25HC or in the presence of T0901317, synthetic liver oxysterol receptor (LXR) agonist, SULT2B1b overexpression had no effect on the regulation of key genes involved in lipid metabolism. Conclusions Our data indicate that sulfation of 25HC by SULT2B1b plays an important role in the maintenance of intracellular lipid homeostasis via the LXR/SREBP-1c signaling pathway in HAECs.

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.

Glucocorticoid-induciblehydroxysteroid sulfotransferase (SULT2-40/41) gene transcription was investigated in primary cultured rat hepatocytes transiently transfected with a series of SULT2-40/41 5Ј-flanking region-luciferase reporter constructs, with emphasis on examining the functional role of an inverted repeat-0 nuclear receptor motif (IR0). Treatment of transfected cultures with any of four glucocorticoids activated luciferase expression from a construct containing 1938 base pairs (bp) of the SULT2-40/41 gene 5Ј-flanking sequence, whereas deletion of bp Ϫ227 to Ϫ158 (containing the IR0 motif) largely abolished the effect. On closer analysis, treatment of hepatocyte cultures with either of the potent glucocorticoids dexamethasone [strong cytochrome P-450 3A (CYP3A) inducer] or triamcinolone acetonide (weak CYP3A inducer) produced dose-dependent increases in luciferase activity when hepatocytes were transiently transfected with a construct containing as little as 158 bp of 5Ј-flanking sequence or containing a mutated IR0 motif. The dexamethasone dose-dependent increase in luciferase activity continued through a dose of 10 Ϫ6 M when the transfected construct contained the IR0 motif, but was maximal at 10 Ϫ7 M when the transfected construct lacked the IR0 motif. In contrast, triamcinolone acetonide-induced luciferase activity was maximal at a dose of 10 Ϫ7 M, irrespective of the presence or absence of the IR0 motif. Coincubation of transfected hepatocytes with 10 Ϫ8 M dexamethasone and the antiglucocorticoid RU486 inhibited luciferase expression. Luciferase induction by the prototypical CYP3A inducer pregnenolone 16␣-carbonitrile was restricted to constructs containing the IR0 motif. These data suggest that glucocorticoid-inducible SULT2-40/41 gene expression occurs through a dual mechanism, whose components possibly involve the glucocorticoid receptor and the pregnane X receptor.The hydroxysteroid sulfotransferases (SULT2) play critical roles in drug metabolism, bile acid detoxication, and carcinogen activation, and in the regulation of intratissue active hormone levels. Therefore, understanding the molecular mechanisms that regulate the expression of this multigene family is important. SULT2 enzymes catalyze the sulfonation of a wide range of sulfate acceptor molecules such as hydroxysteroid hormones, bile acids, aliphatic alcohols, procarcinogens such as 5-hydroxymethylchrysene, and other endogenous and exogenous compounds (Jakoby et al., 1980;Barnes et al., 1989;Ogura et al., 1990b). Depending on the stability of the sulfate ester that is formed, SULT2-catalyzed reactions may culminate in the creation of a polar end product that is amenable to excretion and elimination (detoxication) or in the bioactivation of a procarcinogen to a highly reactive intermediate. Moreover, because sulfated hormones are generally considered to be receptor inactive, alterations in SULT2 gene expression have the potential to shift the balance of intratissue active hormone levels and affect gene expression.In a broad-based...

ABSTRACT:To determine whether the dexamethasone (DEX)-inducible hepatic sulfotransferase gene expression that has been described in the rat is conserved in humans, the effects of DEX treatment on hydroxysteroid sulfotransferase (SULT2A1) and aryl sulfotransferase (SULT1A1) gene expression were investigated in primary cultured human hepatocytes. Hepatocytes were prepared from nontransplantable human livers by collagenase perfusion of the left hepatic lobe, and cultured in Williams' medium E that was supplemented with 0.25 U/ml insulin. As reported in the rat, DEX treatment produced concentration-dependent increases in SULT2A1 mRNA and protein expression, with maximum increases observed at concentrations of DEX that would be expected to activate the pregnane X receptor (PXR) transcription factor. In contrast to the rat, in which DEX-inducible SULT1A1 expression has been demonstrated, SULT1A1 expression in primary cultured human hepatocytes was not measurably increased by DEX. In transient transfections conducted in primary cultured rat hepatocytes, the PXR ligands DEX and pregnenolone-16␣-carbonitrile significantly induced transcription of human and rat SULT2A reporter gene constructs. Cotransfection of either the human or rat SULT2A reporter gene with a PXR dominant negative construct significantly reduced DEX-inducible transcription. These results underscore that while certain features of rat hepatic sulfotransferase gene regulation are conserved in humans, important differences exist across species. The findings also implicate a role for the PXR transcription factor in DEXinducible rat and human SULT2A gene expression.The cytosolic aryl sulfotransferase (SULT1A1 2 ) and hydroxysteroid sulfotransferase (SULT2A1) conjugating enzymes catalyze the transfer of a ϪSO 3 H moiety from the physiological sulfate donor 3Ј-phosphoadenosine-5Ј-phosphosulfate to the appropriate phenolic or hydroxysteroid substrates, respectively (Jakoby et al., 1980). In drug metabolism, sulfate conjugation is recognized as a double-edged sword. As a rule, sulfate conjugates are more polar than the parent substrate and hence, more amenable to excretion and elimination. However, the production of unstable sulfate conjugates can lead to the focused generation of genotoxic species and carcinogen activation.In both rats and humans, SULT1A1 and SULT2A1 enzymes are abundantly expressed in the liver, which is the seat of drug metabolism in mammalian species Falany et al., 1995). Human SULT2A1 is also expressed in the fetal (Parker et al., 1994) and adult adrenal gland (Comer and Falany, 1992), the adult small intestine (Her et al., 1996), and gastric mucosa (Tashiro et al., 2000). Relative to SULT2A1, human SULT1A1 is more extensively expressed in extra-hepatic tissues. SULT1A1 detoxifies common phenolic pharmaceuticals, such as acetaminophen (Larrey et al., 1986) and troglitazone (Honma et al., 2001), and metabolizes the hypotensive and hypertrichotic drug minoxidil to its pharmacologically active form (Falany and Kerl, 1990). The well described g...

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