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Published in final edited form as: Gene. 2006 Oct 20;386(1-2):218–223. doi: 10.1016/j.gene.2006.10.006

Xenobiotic- and Vitamin D-responsive Induction of the Steroid/bile acid-sulfotransferase Sult2A1 in Young and Old Mice: The role of a Gene Enhancer in the Liver Chromatin

Young-Kyo Seo 1,2, Yoon-Tae Chung 1, Soyoung Kim 1,2, Ibtissam Echchgadda 1, Chung S Song 1, Bandana Chatterjee 1,2,*
PMCID: PMC1888572  NIHMSID: NIHMS16206  PMID: 17123747

Abstract

The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D3-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity (Gachon et al, 2006; McElwee et al, 2004). Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month and 20-month old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D3 and the catatoxic synthetic steroid PCN (pregnenolone-16α-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-α (retinoid X receptor) and PXR in mice injected with D3 or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.

Keywords: Aging, Sulfotransferase, Gene Enhancer, Nuclear Receptor, Tissue ChIP

1. Introduction

The xenobiotic-regulated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) play central roles in the induced expression of the enzymes and transporters that mediate the metabolism and clearance of endogenous sterols and steroids and diverse xenobiotics including therapy related drugs (Handschin and Meyer, 2003; Xu et al, 2005). The xenobiotic stress induced by an aberrant metabolome is associated with severe health adversity (Gachon et al, 2006; Green and Takahashi, 2006). Thus, a thorough understanding of the regulatory underpinnings for these enzymes and proteins should be insightful in identifying potential targets amenable to therapeutic modulations. Furthermore, differential phase I/II/III activity profiles between the young and old, including the profiles for drug metabolism and disposition, could arise from the altered expression of steroid- and drug-metabolizing enzymes/transporters during aging. An assessment in this regard is necessary to develop remedial strategies in the elderly. In the present study young and old mice were examined for the induced expression of the phase II sulfotransferase SULT2A1 in the liver upon administration of the synthetic catatoxic steroid PCN (pregnenolone-16α-carbonitrile), which is an agonist ligand for PXR, or vitamin D3, which binds and activates the vitamin D receptor (VDR). Vitamin D3 has a known role in xenobiotic metabolism in addition to its classic function in bone physiology (Makishima et al. 2002; Reschly and Krasowski, 2006; Song et al, 2006). Additionally, we probed the mouse liver chromatin to investigate the association of nuclear receptors and coregulators with a vitamin D- and xenobiotic-responsive enhancer in the Sult2A1 promoter.

SULT2A1 is a sulfo-conjugating enzyme with substrate predilection for hydroxysteroids, bile acids and certain medicinal compounds. The enzyme is expressed at high abundance in the first-pass tissues (liver and intestine) and in the steroidogenic tissue of the adrenal gland. Multiple nuclear receptors including PXR, CAR and VDR can induce SULT2A1 in cultured cells and in vivo in the mouse liver (Song et al, 2001; Sonoda et al, 2002; Echchgadda et al, 2004a; Assem et al, 2004; Maglich et al, 2004; Fang et al, 2005; Seeley et al, 2005; Chatterjee et al, 2005; Song et al, 2006). We had shown earlier that the vitamin D3- and xenobiotic-mediated induction of the mouse Sult2A1 promoter in transfected liver and intestinal cells is dependent upon binding of the corresponding receptors to a 21-base pair inverted repeat (IR0) enhancer (Echchgadda et al, 2004b). Chromatin immunoprecipitation (ChIP) assay showed that the IR0 in the Sult2A1 chromatin was enriched for VDR in the livers of vitamin D3-injected mice, thus validating the enhancer’s role in a tissue context (Song et al, 2006). In the present report we show that aging has no significant influence on the qualitative and quantitative profiles of the Sult2A1 mRNA induction by vitamin D3 or PCN. Also, young and old mice responded similarly to the vitamin D3-/PCN-induced nuclear receptor recruitment at the IR0. This is the first report in which the liver chromatin from young and old mice was analyzed for the functional response to the nuclear receptor signaling.

2. Materials and Methods

2.1 Hormone and drug injections, semi-quantitative RT-PCR, PCR primers

The hormonally active vitamin D (1α,25-dihydroxy vitamin D3, hereon D3) at 50 μg hormone/kg body weight/day or pregnenolone-16α-carbonitrile (PCN) at 50 mg/kg body weight/day was injected intraperitoneally into male mice (C57Bl/6J) for four days. As vehicle, control mice received 0.05M propylene glycol in PBS (for vitamin D3) or DMSO (for PCN). Mice were purchased from the National Institute on Aging (Bethesda); they were handled as per the approved institutional animal care protocol. Total liver RNAs, isolated by Trizol (Invitrogen), were analyzed semi-quantitatively for Sult2A1 mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Reverse transcription of RNAs and PCR amplification of the reverse transcribed cDNAs were as described (Echchgadda et al, 2004b). PCR primers were selected from the N-terminal end of the mouse Sult2A1 cDNA in order to avoid cross reactivity with any other SULT family members. The following primer sets were used: Sult2A1, 5′-GAAGGCATACCTTTT-CCTGCCAT (at +51, forward); 5′GTAACCA-GACACAAGAATATCTCT (at +419, reverse). Gapdh, 5′-GTATTGGGCGCCTGGTCACCAG (at + 121, forward); 5′-CCTTCTCCATGG-TGGTGAAGAC (at + 410, reverse).

2.2 Chromatin immunoprecipitation (ChIP) with liver samples

i) Preparation of solubilized liver chromatin fragments

ChIP was performed on the mouse liver nuclear lysate using the procedure of Sandoval et al, 2004, with minor modifications. Fresh liver (~2 gm) was placed on ice-cold phosphate buffered saline, pH 7.5 (PBS) in a Petri dish, chopped in small pieces (~0.2 gm each), rinsed with fresh PBS and then shaken gently in 1% formaldehyde (in PBS) using an orbital shaker (10 min, room temp.). Cross linking was stopped by adding 125mM glycine, pH 3.0 (10 min, room temperature). The fixed tissue, rinsed once (ice-cold PBS), was incubated in 2 volumes of ice-cold lysis buffer (5mM HEPES pH8.0, KCl 85mM, NP40 0.5% and a protease inhibitor cocktail from Sigma at 1:1000 dilution) for 30 min at 0°C. The lysed tissue was homogenized for 30 seconds on ice (Polytron™ homogenizer, 10 mm diameter), incubated further on ice (15 min) and the homogenate was centrifuged (3500× g, 5 min) to pellet nuclei. The nuclei were lysed with 10 mM EDTA, 1% SDS, 50mM TrisHCl, pH 8.1 at 1:1 (initial tissue weight/volume). The lysate was incubated on ice (10 min), spun quickly (4°C) and the supernatant was sonicated using 3 separate sonication bursts at 30% amplitude (Vibra-Cell VCX-500 sonicator from Sonics and Materials). Each burst lasted 25 sec (at a pulse-grade 7) with a 2-minute resting interval in-between. The sonicated and centrifuged samples (4°C, 10 min, 13,000 rpm) were analyzed on a 1% agarose gel to ensure a 300–800 base pair size range of the fragmented DNAs. The supernatant was diluted 10-fold in a buffer containing the protease inhibitor cocktail and the DNA concentration was measured at OD260.

ii) Specific immunoprecipitation of the chromatin DNA and PCR amplification

Immunoprecipitation of the solubilized chromatin, recovery of the cross-linked protein-DNA complex, reversal of the cross link, and recovery of the immunoprecipitated DNA were carried out as described (Song et al, 2006). All antibodies were from the Santa Cruz Biotechnology (Santa Cruz, CA). Briefly, the immunoprecipitated cross-linked complexes were incubated with protein A-agarose (Upstate Biotechnology, Lake Placid, NY), washed sequentially with low salt buffer, high salt buffer and LiCl buffer and eluted from the protein A-agarose bead. After reversing the cross-linking, DNA was isolated from proteinase K-digested samples and PCR was performed to amplify the mouse Sult2A1 promoter region that spans the IR0 enhancer. The denatured template (100 ng) was amplified for 33 cycles (95°C, 30 sec; 55°C, 30 sec; 72°C, 40 sec) in the presence of: 5′-GCATTTCTATGTCCTATTAC (forward primer at −231) and 5′-GATATGATATGGCAGG-AAAAGGT (reverse primer at + 81).

3. Results

3.1. Induction of Sult2A1 mRNAs and the IR0 enhancer in the mouse liver chromatin

Sult2A1 mRNAs were induced in the mouse liver upon the administration of 1,25 (OH)2D3 (D3) and concomitantly, the IR0 enhancer in the Sult2A1 chromatin was enriched for VDR, RXR-α (the retinoid X receptor), RNA polymerase II and the p160 coactivator SRC-1, as shown in Figure 1. Sult2A1 mRNAs were induced ~3-fold in RT-PCR analysis when young-adult male mice (4-month-old) were injected with D3 (Fig. 1A). Gapdh mRNA expression was the normalization control. To determine fold induction, the amplified DNA bands of the RT-PCR assay corresponding to the control and experimental mice were quantified using a Gel documentation system (Gel Doc™ EQ, Bio-Rad, Hercules, CA). A standard curve was generated from a plot of the PCR band density (Y axis) against the volume of the reverse transcribed reaction mixture used in the PCR amplification (X-axis). The X axis parameter is proportional to the level of Sult2A1 mRNAs in the reaction mixture. The relative levels of Sult2A1 mRNAs in the hormone-injected and control mice were determined from the linear range of the standard curve and fold induction was calculated. The RT-PCR data is in line with our earlier Northern blot results, which showed 3.8 to 6-fold induction of Sult2A1 mRNAs in D3-injected mice (Chatterjee et al, 2005; Song et al, 2006). Animal-to animal variation can explain the 3- to 6-fold induction range.

Figure 1. Sult2A1 gene induction in the liver of vitamin D3-injected mice.

Figure 1

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1A) Induction of Sult2A1 mRNAs as assayed by semi-quantitative RT-PCR. 4-month-old mice were injected either with vehicle (0.05M propylene glycol) or D3, using two mice in each treatment group. 1B) Activity of the IR0 enhancer to recruit VDR, RXR-α, RNA polymerase II (pol II), SRC-1, SRC-2 and SRC-3 in the D3-injected mice. In tissue ChIP assay, IR0-containing liver chromatin fragments of the mouse Sult2A1 promoter were immunoprecipitated with the test antibodies and the specifically immunoprecipitated DNAs were analyzed on an agarose gel subsequent to PCR amplification. The COX-2 antibody served as a negative control.

A direct role of VDR in Sult2A1 induction was demonstrated by immmunoprecipitation (IP) of solubilized chromatin fragments from the livers of mice with or without the hormone treatment (Fig. 1B). Hormone injection caused significant enrichment of the immunoprecipitable VDR at the IR0-spanning Sult2A1 chromatin. The retinoid X receptor (RXR-α) was also enriched at the IR0 in a D3-dependent manner. RXR-α is a heterodimer partner of VDR and thus binds to VDR target genes at the vitamin D response element. Specificity of the receptor association with the IR0 is evidenced by the lack of a PCR signal in the lanes corresponding to chromatin IP with the Cox-2 antibody.

The D3-responsive transcriptional induction paralleled recruitment of RNA polymerase II to the target IR0 site (Fig. 1B). Furthermore, among the p160 coactivators only SRC-1 showed enrichment at the IR0 in response to D3. SRC-2 and SRC-3 did not show hormone-dependent enrichment at IR0. The weak signals observed in the absence of the hormone injection are likely due to Sult2A1 expression promoted by the normally circulating D3. The enrichment of SRC-1 at the IR0 appears to be specifically responsive to the acute increase in the D3 level. However, our earlier ChIP data on the human SULT2A1 chromatin showed that upon D3 treatment of HepG2 human hepatoma cells, both SRC-1 and SRC-2 were inducibly associated with the vitamin D response element that we had identified in the human SULT2A1 gene. On the other hand, SRC-3 was not detected at this site either basally, or in D3-treated cells (Song et al, 2006).

3.2. Aging and vitamin D3 responsiveness of the mouse liver chromatin

Young and old mice responded similarly for Sult2A1 mRNA expression and for VDR and RXR-α recruitment at the IR0 upon D3 injection (Figure 2). Thus, the livers of 4-month and 20-month old mice showed ~ 3-fold and ~ 2-fold induction of Sult2A1 mRNAs, respectively in RT-PCR assay (Fig. 2A). The PCR signals in 20-month-old mouse livers are more intense than those in 4-month-old animals. This increased signal intensity is in line with a higher expression of Sult2A1 mRNAs during aging (Echchgadda et al, 2004a).

Figure 2. Vitamin D3 responsiveness of the Sult2A1 gene in young and old mice.

Figure 2

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2A) RT-PCR analysis of Sult2A1 mRNAs in the livers of vitamin D3 –injected 4 month and 20 month old mice. The numbers at the top in the D3-treated lanes indicate fold increase of Sult2A1 mRNAs in the young (3-fold) and old (2-fold) compared to vehicle treatment. The fold increase was calculated from the scanned gel bands as described under Results. 2B) Immunoprecipitation of solubilized liver chromatin fragments to assay for the enrichment of VDR and RXR-α at the IR0 in vitamin D3-injected 4 month and 20 month old mice. The COX-2 antibody was a negative control.

Chromatin IP showed that young and old mice were almost equally effective in the D3-mediated enrichment of VDR at IR0. Likewise, the extent of D3-responsive RXR-α occupancy at the IR0 was similar in the young and old mice. The specificity of the liver-directed ChIP assay was evident from the absence of PCR signals in lanes where the Cox-2 antibody was used to immunoprecipitate chromatin fragments. These results suggest that both the pre-transcriptional and transcriptional responses of the Sult2A1 gene to the VDR-induced signaling remain unaltered during physiological aging.

3.3. Xenobiotic-mediated induction of the Sult2A1 gene

The effect of aging on responses to the xenobiotic signaling was explored in PCN-injected mice (Figure 3). The catatoxic steroid PCN, which is a derivative of the C21 steroid pregnenolone, renders protection to animals against drugs and toxins by stimulating xenobiotic metabolism (Selye, 1971). PCN is also a glucocorticoid antagonist. The catatoxic activity of PCN is based on its ligand agonist activity toward PXR, thus stimulating the expression of the enzymes and proteins involved in the steroid and xenobiotic metabolism. Therefore, PCN-mediated gene expression can be taken as a measure of drug responsiveness. Sult2A1 mRNAs were induced by PCN to similar extents, i.e. 3-fold versus 2.5-fold in the livers of 4-month- and 20-month-old mice, respectively (Fig. 3A). The mRNA induction is due to promoter activation since we had shown earlier that in PXR cotransfected HepG2 cells, luciferase expression driven by the mouse Sult2A1 promoter increased several fold by the activated PXR, and this induction was mediated through the IR0 enhancer (Echchgadda et al, 2004b).

Figure 3. Sult2A1 gene induction in PCN-injected young and old mice.

Figure 3

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3A) RT-PCR analysis of the Sult2A1 mRNA induction in the livers of 4-month and 20-month old PCN-injected mice. Numbers at the top indicate the fold induction of Sult2A1 mRNAs after PCN injection compared to the vehicle control. 3B) ChIP analysis of liver chromatin from young and old mice to determine the activity of IR0 to recruit PXR and RXR-α in response to PCN injection.

The IR0’s activity to recruit PXR and its heterodimer partner RXR-α in young and old mice was investigated using chromatin IP (Fig. 3B). Higher amounts of PXR and RXR-α specifically associated with the IR0-spanning Sult2A1 chromatin in the PCN-injected mouse liver compared to the control liver. Aging did not alter the IR0 activity in this regard. Fig. 2 and Fig. 3, taken together, lead us to conclude that the responses of Sult2A1 to the vitamin D3 and PCN signaling are refractory to the age-associated changes in the metabolic and endocrine milieu of the liver.

4. Discussion

It is of significant interest to know how aging might modulate xenobiotic metabolism, since extended life span is known to be associated with increased expression of the enzymes and proteins that are involved in the metabolism of steroids, drugs and other xenobiotics (McElwee et al, 2004; Amador-Noguez et al, 2004). Transcription profiling has revealed that as in development, physiological aging is associated with differential expression of a wide variety of genes that have important metabolic relevance (Park & Prola, 2005; Darlington, 2004). For example, after age 40 the frontal cortical tissue in humans shows reduced expression of a set of genes involved in synaptic plasticity, vesicular transport and mitochondrial function, whereas genes with roles in the DNA repair, stress response and anti-oxidative response were up regulated (Lu et al, 2004). Nevertheless, specific chromatin events leading to the age-related changes in the transcriptional activity of specific genes, including those for drug-metabolizing enzymes, are not known. The use of tissue-directed ChIP to probe the transcriptional response of the Sult2A1 gene in young and old mice is a useful advance in this regard. We show that young and aged tissues responded similarly with respect to the hormone- (vitamin D3) and drug- (PCN) mediated induction of Sult2A1. Additionally, our results show that an IR0 gene enhancer in the liver chromatin, which directs the vitamin D3 and PCN induction of Sult2A1, is active in recruiting VDR, RXR-α and PXR to almost equal extents in young and old animals in response to the hormone (D3) or drug (PCN) challenge.

The age effect of the nuclear receptor CAR on the induction of the Sult2A1 gene was not examined in the current study. Nevertheless, the CAR-regulated hepatocyte proliferation and the induction of a Forkhead Box transcription factor in the liver were not altered during aging (Ledda-Columbano et al, 2004). Thus, it is likely that the regulation of Sult2A1 by CAR is also unaffected by age. It is important to note, however, that a lack of CAR expression is associated with epileptic seizure in young mice and accelerated aging in mice older than 9 months (Gachon et al, 2006). Thus the aging process appears to affect a selected set of gene networks that are regulated by the CAR-mediated nuclear signaling.

ChIP provides a biochemical means to capture a snapshot of proteins that are associated with specific chromatin regions in intact cells, and its application on tissue samples is useful in gaining insights into the transcriptional regulation of genes in a physiologically relevant setting. For example, metamorphosis related differential coregulator recruitment to specific genes has been studied by the ChIP analysis of tadpole tissues; zebrafish development has been examined using ChIP on embryonic tissues; ChIP has also been used on the mouse liver chromatin to analyze specific enhancer complexes (Parrazis et al, 2001; Chaya and Zaret, 2003; Havis et al, 2003; Havis et al, 2006). The present report further extends the usefulness of tissue-directed ChIP to study chromatin events during aging.

SULT2A1 is member of the cytosolic sulfotransferase (SULT) family that includes 47 individual SULTs (Blanchard et al, 2004). Bile acid detoxification and clearance in humans is mediated exclusively through SULT2A1-directed sulfonation (Palmer, 1971). Thus, SULT2A1 activity plays an important role in cholesterol and bile acid metabolism. Aging is associated with marked up regulation of the Sult2A1 gene in the rodent liver – a regulatory process that is explained, in part, by the loss of androgen receptor expression in the aging liver (Echchgadda et al, 2004a). Hormone- and age-regulated changes in SULT2A1 expression may alter cholesterol homeostasis due to altered bile acid clearance; changes in SULT2A1 expression may also have impacts as to how steroids, drugs and xenobiotics are metabolized. Therefore, illuminating the mechanism underlying SULT2A1 expression during aging is important in order to assess the physiological and clinical impacts of the SULT2A1 activity. In view of the exclusive role of SULT2A1 in bile acid clearance; its role for other endobiotic and xenobiotic substrates in first-pass metabolism; its age-dependent differential expression; and its induction by multiple nuclear receptors, a detailed knowledge of the genomic events at the SULT2A1 chromatin has the potential to provide insightful clues to the effective management of diseases related to aberrant steroid and drug metabolism.

Acknowledgments

This work was supported by NIH grants AG-19660 & AG-10486; a Merit-Review grant and a Senior Research Career Scientist Award from the Department of Veterans Affairs; and a grant from Philip Morris USA, Inc. and Philip Morris International. We thank John Isidro De La Cruz for skilled technical assistance.

Abbreviations

PCN

pregnenolone-16α-carbonitrile

D3

1α,25-dihydroxy vitamin D3

IR0

inverted repeat-zero spacer

VDR

vitamin D receptor

PXR

pregnane X receptor

RXRα

retinoid X receptor α

CAR

constitutive androstane receptor

ChIP

chromatin immunoprecipitation

RT

reverse transcription

PCR

polymerase chain reaction

Footnotes

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