Gene regulation for the senescence marker protein DHEA-sulfotransferase by the xenobiotic-activated nuclear pregnane X receptor (PXR)

The authors, and all other members of the Roy-laboratory and Chatterjee-laboratory, pay tribute to Prof. Arun K. Roy, an imaginative scientist, inspiring mentor and a vibrant friend and colleague.1
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Abstract

Dehydroepiandrosterone (DHEA)-sulfotransferase (SULT2A1) is a phase II metabolizing/detoxifying enzyme with substrate preference for physiological hydroxysteroids, diverse drugs and other xenobiotics. The first-pass tissues (liver and intestine) express SULT2A1 at high levels. In senescent male rodents, Sult2A1 gene transcription in the liver is markedly enhanced and calorie restriction retards this increase. Age-associated loss of the liver expression of androgen receptor in part explains the up-regulation of Sult2A1 expression at late life, since androgen receptor is a negative regulator of this gene. In line with its role in xenobiotic metabolism, the Sult2A1 gene is induced by the pregnane X receptor (PXR). PXR is a xenosensing nuclear receptor that is activated by endobiotic (natural steroids) and xenobiotic (therapeutic drugs and environmental chemicals) molecules. An inverted-repeat arrangement (IR0) of the consensus half site binding sequence for nuclear receptors mediates the xenobiotic induction of the Sult2A1 promoter. The IR0 element is a specific binding site for PXR and its heterodimer partner retinoid X receptor (RXR-α) and it directs PXR-mediated induction of a heterologous promoter. In contrast to the loss of androgen receptor expression, PXR and RXR-α mRNA expression is invariant during aging. Repression by the androgen receptor and induction by PXR may act coordinately to cause the senescence associated and xenobiotic mediated stimulation of Sult2A1 transcription. Increased Sult2A1 expression appears to be an adaptive response to ensure optimal metabolism of Sult2A1 substrates at old age.

Introduction

Normal aging reflects a time-dependent progressive decline in the vitality of living beings caused by functional changes at tissue, cell and molecular levels. A large body of evidence shows that genes and their transcriptional profiling play important roles in regulating life span and the rate of aging (Longo and Finch, 2003, Csoka et al., 2004, Lee et al., 2004, Lu et al., 2004). Aging is often associated with altered responses to the epigenetic influences of endogenous and exogenous molecules. A general aging-related decline in drug metabolism frequently is the cause for adverse reactions to many therapy related drugs in the elderly. Decreased expression of a number of drug-metabolizing enzymes, as found in animal studies (Galinsky et al., 1986, Dhir and Shapiro, 2003), may in part be responsible for the increased incidences of drug toxicity among the aged. In contrast, however, several enzymes involved in steroid/drug/xenobiotic metabolism are expressed at higher levels in old animals compared to young-adults. In this case, the stimulated enzyme expression likely manifests an adaptive response to life-long exposure to steroids, drugs and environmental chemicals. The conjugating enzyme dehydroepiandrosterone (DHEA)-sulfotransferase is one such example, for which the male rodent liver displays marked transcriptional stimulation during aging. In this article we present our results on the induction of this sulfotransferase during aging and in response to xenobiotics. We also review the role of nuclear receptors in the regulation of xenobiotic metabolism, and highlight the potential importance of DHEA-sulfotransferase and the sulfonation pathway in drug–drug, drug–food and drug–herb interactions.

DHEA-sulfotransferase (SULT2A1; EC 2.8.2.2) is a cytosolic sulfo-conjugating enzyme expressed at high abundance in the first-pass enterohepatic tissues and in the steroidogenic adrenal tissue (Weinshilboum et al., 1997, Falany, 1997). As a broad specificity phase II enzyme, the SULT2A1 activity in the enterohepatic tissues protects cells against toxic build-up of steroids, clinically active drugs and environmental chemicals. SULT2A1 substrates include physiological hydroxysteroids (dehydroepiandrosterone, testosterone, estrogen, pregnenolone and bile acids/bile salts) (Chatterjee et al., 1994, Falany, 1997, Song et al., 2001, Strott, 2002), and diverse lipophilic hydroxylated xenochemicals, examples being: environmental estrogens, hydroxy-tamoxifen (an anti-breast cancer drug), budesonide (an anti-inflammatory glucocorticoid useful in the treatment of asthma and Crohn's disease) and environmental pollutants containing benzylic alcohols (Glatt, 1997, Pai et al., 2002, Meloche et al., 2002, Kim et al., 2004). SULT2A1 and all other members of the sulfotransferase family utilize 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfate-donating cofactor. The synthesis of PAPS from ATP and inorganic sulfate is catalyzed by PAPS synthetase. The high polarity of the sulfate group facilitates solubilization, intracellular transport and ultimate excretion of the sulfated metabolites. Steroids and xenobiotics are also metabolized by the phase I cytochrome P450 (CYP) monooxygenases (Lewis, 2004). In some instances, the hydroxyl groups produced de novo by CYP enzymes are targeted for further metabolism through sulfonation. A heightened importance of SULT2A1 as a cytoprotectant would be expected under conditions when CYPs or other conjugating transferases (glucuronosyltransferase; glutathione-S-transferase (GST) and others) are functionally impaired due to genetic, dietary and environmental factors.

Early studies in our laboratory on the age-dependent changes of gene expression in rodents had led to the identification of an androgen-repressible 32-kDa senescence marker protein-2 (SMP-2). SMP-2 is present at a low to almost non-detectable level in post-pubertal, young male livers; however, its mRNA and protein expression rises steadily with increasing age and by late senescence (older than 20 months of age) the SMP-2 level in the male liver is more than an order of magnitude higher than that in 6-month-old rats (Chatterjee et al., 1981, Chatterjee et al., 1987, Chatterjee et al., 1990, Song et al., 1990). Calorie restriction reverses the age-associated rise in SMP-2 expression (Chatterjee et al., 1989). Cloning of the cDNAs encoding the members of the SULT family, and characterization of the enzymatic function of the recombinant SMP-2 helped establish that this senescence marker protein is identical to SULT2A1 (Ogura et al., 1990, Chatterjee et al., 1994, Kong and Fei, 1994). SULT2A1/SMP-2 expression is transcriptionally repressed by androgens (Chatterjee et al., 1987, Demyan et al., 1992, Song et al., 1998). Due to androgen repressibility, Sult2A1 is expressed in the female liver all throughout life. We showed earlier that androgen receptor expression in the liver declines steadily with increasing age, precipitating down to a non-detectable level by late senescence (Song et al., 1991, Supakar et al., 1993, Roy et al., 1999). Loss of androgen receptor expression and the consequent release of androgenic repression on the Sult2A1 gene likely explains, at least in part, the age-associated increased expression of this senescence marker protein. As an example of the substrate-regulated activation of enzyme expression, SULT2A1 transcription is induced by bile acids (Song et al., 2001) and by diverse xenobiotic agents (Runge-Morris et al., 1999, Duanmu et al., 2002, Echchgadda et al., 2002, Echchgadda et al., 2003, Echchgadda et al., 2004, Sonoda et al., 2002).

Mammalian gene expression in response to xenobiotic challenge is mediated by two xenobiotic-activated transcription factors—the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). PXR and CAR are members of the nuclear receptor superfamily, and like most other non-steroid nuclear receptors, the transcription regulatory function of PXR and CAR is dependent upon obligatory heterodimerization with the 9-cis retinoic acid receptor known as the retinoid X receptor (RXR-α). PXR ligand agonists include natural steroids (C21, C20 and C19) and various xenochemicals including prescription drugs, pesticides, endocrine disruptors and environmental contaminants such as polychlorinated biphenols (Kliewer, 2003). Examples of common prescription drugs that activate PXR include: rifampicin (antibiotic); dexamethasone (anti-inflammatory); cyproterone acetate (antiandrogen); mifepristone (known as RU486, the abortifacient); lovastatin (anti-hypercholesterolemic); tamoxifen and paclitaxel (anticancer); troglitazone (antidiabetic), ritonavir (anti-HIV protease inhibitor), clotrimazole (antimycotic) and hyperforin (antidepressant; a constituent of the herbal medicine St. John's wort) (Kliewer et al., 2002). High levels of the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) also activate PXR (Xie et al., 2001, Schuetz et al., 2001). In comparison, the repertoire of chemicals known to activate CAR is less broad. CAR activators include phenobarbital-like molecules, certain pesticides and a limited set of drugs (Chawla et al., 2001). The large volume and the smooth, elliptical shape of the ligand-binding pocket of PXR is thought to facilitate its binding to disparate chemicals (Watkins et al., 2001).

More than 50% of all marketed medicines are metabolized by CYP3A4 in the human liver. Induction of CYP3A4 gene transcription by the drug-activated PXR (and to a lesser extent by CAR), forms the metabolic basis for drug–drug interaction. In situations involving intake of multiple prescribed drugs (a scenario all too common in modern-era medicine), induction of CYP3A4 activity by one drug would accelerate the metabolism of a second drug, thus strengthening or reducing drug potency. SULT mediated sulfonation acting independently of CYP3A4 may be equally important in drug/steroid metabolism and in drug–drug interaction. Indeed, it was shown in mouse experiments that bile acid detoxification utilizes a CAR-activated, SULT2A1-dependent sulfonation pathway, completely bypassing the CYP3A dependent metabolic cascade (Saini et al., 2004). Ligand-activated PXR also induces the PAPS-synthesizing enzyme and phenol sulfotransferases (Xie et al., 2001). With the emergence of recent experimental evidences for the importance of sulfonation induced steroid and drug metabolism, attention is increasingly being directed to understanding the regulation of sulfotransferase genes and contribution of the sulfonation pathway to pharmacokinetics and to drug–drug interactions.

PXR and CAR stimulate SULT2A1 gene expression in the liver and intestine and in cultured cells derived from these tissues (Echchgadda et al., 2002, Echchgadda et al., 2003, Echchgadda et al., 2004, Sonoda et al., 2002, Saini et al., 2004, Assem et al., 2004). Mouse livers inactivated for PXR or CAR fail to be induced for Sult2A1 (Sonoda et al., 2002, Assem et al., 2004). In transgenic mouse livers activation of CAR caused induction of the Sult2A1 gene (Saini et al., 2004). Sult2A1 may be especially important in protecting the hepatobiliary system against cholestasis induced by the secondary bile acid lithocholic acid, since sulfonation is the major route for LCA metabolism in humans and SULT2A1 is the exclusive enzyme for LCA sulfo-conjugation (Radominska et al., 1990). Sulfated steroids and sulfated bile acids are exported from the liver with the help of MRP-4, a transporter protein that is highly induced in the cholestatic liver (Assem et al., 2004). LCA is a low-affinity PXR ligand and PXR-null mice develop cholestasis upon LCA administration, presumably due to the lack of Sult2A1 induction (Xie et al., 2001, Staudinger et al., 2001). Indeed, the direct evidence for a protective role of Sult2A1 against LCA-induced hepatic toxicity has recently come from the studies by Kitada et al. (2003).

In this article we show that the xenobiotic induction of the rodent Sult2A1 is a sequel to the binding of the ligand-activated PXR to a palindromic, inverted repeat (IR0) DNA element in the proximal promoter of this gene. Our studies on live-cell imaging showed uniform distributions of PXR and RXR-α over the entire nucleus in the absence of a PXR ligand, while dexamethasone, a PXR ligand, induced migration of a fraction of the nuclear PXR to intranuclear foci. We speculate that these foci may represent PML bodies that are the presumptive sequestering sites for transcription-promoting regulatory factors, and that the ligand-induced focal migration of PXR may represent an early step in the transcription of Sult2A1 and other PXR target genes. PXR and RXR-α mRNA expression in the mouse liver remains invariant during aging unlike the androgen receptor, which shows age-associated decline and ultimate loss of expression. The opposing regulation of Sult2A1 by the androgen receptor (a negative regulator) and PXR (a positive regulator) may act coordinately to orchestrate increased gene transcription for Sult2A1 during aging and xenobiotic challenge.

Section snippets

Animals, drug treatment, RNA analysis and Western blot

For aging studies, Fischer 344 male rats and C57 black male mice were purchased from the colony maintained by the National Institute on Aging. The liver samples from calorie restricted and ad libitum (AL) fed Fischer 344 male rats were from the calorie restriction colony at the University of Texas Health Science Center at San Antonio. To study xenobiotic induction, C57 black male mice (∼8-week-old) were given a single intraperitoneal injection of pregnenolone 16α-carbonitrile (PCN) at 0.4 mg/g

Enhanced Sult2A1 expression at late senescence and its reversal by calorie restriction

The age-associated up-regulation of Sult2A1 expression in the liver is shown in Fig. 1. As evident from the Northern blot in Fig. 1A, Sult2A1 mRNAs are expressed at very low levels in 6-, 12- and 18-month-old male rats. However, by 24 months of age, a marked rise in Sult2A1 mRNA expression is observed, and the Sult2A1 mRNA level increased further at 27 months. As in the rat, Sult2A1 mRNAs in the mouse liver also increased steadily during aging as shown in the semi-quantitative RT-PCR assay (

Discussion

The central role of nuclear receptors in drug and steroid metabolism is amply evident from the findings that several members of this receptor family play integral roles in the basal and induced expression of the phase I and phase II enzymes and transporter proteins (Chawla et al., 2001, Kliewer, 2003). Uptake of nutrients/drugs/xenotoxicants, their enzymatic conversions and export out of cells and the elimination of metabolites from the body are coordinated through transporter proteins, phase I

Perspectives

Single nucleotide polymorphism (SNP) of human SULT2A1 has been identified for Caucasian-American and African-American subjects (Thomae et al., 2002). Several of the SNPs residing within the protein-coding sequence are associated with reduced expression and activity of SULT2A1 in transfected cells. Ethnic group-specific polymorphism in the SULT2A1 gene has also been noted (Thomae et al., 2002). For human PXR, extensive genetic polymorphism within the Caucasian, African and African-American

Acknowledgements

Supported by NIH (R01AG-10486 and T32-AG 00165) and the Philip Morris USA. B.C. is a VA Senior Research Career Scientist. We thank Srikanth Kota for technical help, Jason Lu for constructing CFP-PXR and Gilbert Torralva for help with graphics.

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    The senescence marker protein was first identified in the early 1980s when one of us (B.C.) was running experiments with Prof. Roy and navigating through an uncharted research area, namely differential gene expression during aging. Prof. Roy's legacy will continue through the work of his students, fellows, collaborators and colleagues—all of whom gained immeasurably from his vision and wisdom.

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