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Molecular Endocrinology, doi:10.1210/me.2005-0428
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Molecular Endocrinology 20 (4): 795-808
Copyright © 2006 by The Endocrine Society

An Essential Role of the CAAT/Enhancer Binding Protein-{alpha} in the Vitamin D-Induced Expression of the Human Steroid/Bile Acid-Sulfotransferase (SULT2A1)

Chung S. Song, Ibtissam Echchgadda, Young-Kyo Seo, Taesung Oh, Soyoung Kim, Sung-A Kim, Sunghwan Cho, Liheng Shi and Bandana Chatterjee

Department of Molecular Medicine/Institute of Biotechnology (C.S.S., I.E., Y.-K.S., T.O., S.K., S.-A.K., S.C., L.S., B.C.), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245; and South Texas Veterans Health Care System (S.K., S.C., B.C.), Audie L. Murphy Veterans Affairs Hospital, San Antonio, Texas 78229

Address all correspondence and requests for reprints to: Bandana Chatterjee, Ph.D., Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245. E-mail: chatterjee{at}uthscsa.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The vitamin D receptor (VDR) regulates steroid and drug metabolism by inducing the genes encoding phase I and phase II enzymes. SULT2A1 is a liver- and intestine-expressed sulfo-conjugating enzyme that converts the alcohol-OH of neutral steroids, bile acids, and drugs to water-soluble sulfated metabolites. 1{alpha},25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces SULT2A1 gene transcription after the recruitment of VDR to the vitamin D-responsive chromatin region of SULT2A1. A composite element in human SULT2A1 directs the 1,25-(OH)2D3-mediated induction of natural and heterologous promoters. This element combines a VDR/retinoid X receptor-{alpha}-binding site [vitamin D response element (VDRE)], which is an imperfect inverted repeat 2 of AGCTCA, and a CAAT/enhancer binding protein (C/EBP)-binding site located 9 bp downstream to VDRE. The binding sites were identified by EMSA, antibody supershift, and deoxyribonuclease I footprinting. C/EBP-{alpha} at the composite element plays an essential role in the VDR regulation of SULT2A1, because 1) induction was lost for promoters with inactivating mutations at the VDRE or C/EBP element; 2) SULT2A1 induction by 1,25-(OH)2D3 in C/EBP-{alpha}-deficient cells required the expression of cotransfected C/EBP-{alpha}; and 3) C/EBP-ß did not substitute for C/EBP-{alpha} in this regulation. VDR and C/EBP-{alpha} were recruited concurrently to the composite element along with the coactivators p300, steroid receptor coactivator 1 (SRC-1), and SRC-2, but not SRC-3. VDR and C/EBP-{alpha} associated endogenously as a DNA-dependent, coimmunoprecipitable complex, which was detected at a markedly higher level in 1,25-(OH)2D3-treated cells. These results provide the first example of the essential role of the interaction in cis between C/EBP-{alpha} and VDR in directing 1,25-(OH)2D3-induced expression of a VDR target gene.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE METABOLISM, DETOXIFICATION, and disposition of hormones, nutrients, and diverse xenobiotics including medicinal drugs require coordinate activities of a network of enzymes and transporters in first-pass tissues, most notably in the liver and intestine. Several nuclear receptors, including the vitamin D receptor (VDR), regulate the transcriptional expression of these enzymes and proteins. Transactivation by VDR normally requires direct binding of the agonist-activated receptor and its heterodimer partner retinoid X receptor-{alpha} (RXR-{alpha}, the 9-cis-retinoic acid receptor) to the vitamin D-response element in target genes. VDR has pleiotropic influences on cells as evidenced by its key role in calcium and phosphate homeostasis; its ability to induce differentiation, apoptosis, and growth inhibition of epithelial cells; and its regulation of steroid and drug metabolism by stimulating gene transcription for phase I and phase II enzymes (1, 2, 3, 4, 5, 6, 7). Induction of the genes encoding phase I cytochrome P450 (CYP) monooxygenases by the highly cytotoxic lithocholic acid (LCA), which is an agonist ligand for VDR, further underscores the importance of the VDR-regulated signaling in metabolic detoxification (8, 9). SULT2A1, the gene encoding the phase II steroid and bile acid sulfotransferase (commonly known as dehydroepiandrosterone sulfotransferase), is also induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. A VDR-binding element in the rodent Sult2A1 gene was previously characterized and was shown to confer vitamin D response to a heterologous promoter (5, 9).

SULT2A1 is a mammalian cytosolic sulfotransferase that catalyzes sulfate esterification of the alcohol-OH of neutral steroids, amphipathic sterol acids (known as bile acids), and various xenobiotics, including common therapy-related drugs. SULT2A1 expression is most abundant in the liver and intestine and in the steroidogenic adrenal cortex (10, 11, 12, 13). SULT2A1 substrates include a subgroup of steroids (dehydroepiandrosterone; testosterone, dihydrotestosterone; androstanediol; pregnenolone; and 17ß-estradiol) and primary and secondary bile acids, which are produced in the liver as the end catabolites of cholesterol. Facile elimination of the polar, water-soluble sulfated products as excreted metabolites prevents toxic accumulation of these molecules within cells. In humans, sulfonation by SULT2A1 rather than hydroxylation by CYP enzymes appears to be the preferential route directing bile acid detoxification and clearance (14). SULT2A1 also catalyzes sulfate group transfer to the antiestrogens hydroxytamoxifen (a breast cancer drug) and raloxifene (an osteoporosis drug); the glucocorticoid agonist budenoside (an antiinflammation drug for Crohn’s disease); and various environmental estrogens (9, 15, 16, 17, 18, 19). The phase II role of SULT2A1 in the metabolism of such a broad range of endobiotics and xenobiotics underscores the importance of this enzyme in normal physiology and in pathological settings. Several nuclear receptors regulate SULT2A1 gene transcription through their direct roles at cognate hormone response elements. The receptors for vitamin D (VDR); glucocorticoids [glucocorticoid receptor (GR)]; fatty acids (peroxisome proliferator-activated receptor-{alpha}); bile acids [farnesoid X receptor (FXR)]; xenobiotics [pregnane X receptor (PXR) and constitutive androstane receptor (CAR)], as well as the orphan nuclear receptors steroidogenic factor 1 (SF1) and estrogen-related receptor-{alpha} induce SULT2A1 expression, whereas the androgen receptor is a negative regulator of this gene (12, 13, 16, 20, 21, 22, 23, 24, 25, 26).

The goal of the present study was to investigate the mechanism underlying the human SULT2A1 gene induction by 1,25-(OH)2D3. We have characterized a vitamin D-responsive region in the upstream promoter and explored how a CAAT/enhancer binding protein (C/EBP) element that resides in close proximity to a VDR-binding element would influence the SULT2A1 promoter function. C/EBP-{alpha} is a basic leucine zipper protein of the CAAT/enhancer binding (C/EBP) family of transcription factors; it is expressed at high levels in terminally differentiated cells, most notably in the hepatocytes, enterocytes, and adipocytes (27); and it plays a key role in many aspects of the liver and intestinal biology, including the detoxification function of these tissues (28). We show that, unlike the rodent gene, the 1,25-(OH)2D3-induced expression of human SULT2A1 involves complete functional interdependence between VDR and C/EBP-{alpha}. Furthermore, the closely related C/EBP-ß cannot substitute for the essential role of C/EBP-{alpha} in the VDR-mediated regulation of human SULT2A1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of Endogenous Sult2A1 mRNAs by 1,25(OH)2D3 and Recruitment of VDR and RXR-{alpha} to the Hormone-Responsive Region
Previously we had shown that 1,25-(OH)2D3 can induce SULT2A1 mRNA and protein expression in HepG2 hepatoma cells and activated the corresponding human, mouse, and rat promoters in liver and intestinal cells (5). This induction also occurs in vivo in the mouse liver, because Northern blot assay of liver mRNAs from hormone- and vehicle-injected mice showed that Sult2A1 mRNAs increased 3.4-fold by 1,25-(OH)2D3 and 2.8-fold by the VDR agonist EB1089 (Fig. 1Go). The phase I Cyp3A11 was induced by 1,25-(OH)2D3 (7.7-fold) and EB1089 (9.0-fold) as well, as reported previously (4).


Figure 1
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  		Fig. 1. Induction of Sult2A1 and Cyp3A11 mRNAs in Mouse Livers by 1,25-(OH)2D3 and EB1089

Northern blots of liver mRNAs from three different treatment groups of mice (two mice in each group) are shown. Vehicle: 0.05 M propylene glycol; EB1089: 1,25-(OH)2D3 agonist. Signal in each lane was normalized to constant expression of GAPDH mRNAs, and the signals for the two lanes from each treatment group were averaged. Fold inductions were relative to the vehicle-treated group. Ind., Induction; Veh, vehicle.

 
The transfected –303, –267, and –211 SULT2A1 promoter fragments were induced by 1,25-(OH)2D3, whereas the –128 promoter failed to be induced. Thus, a vitamin D-response region maps to an area within the –211 to –128 sequence (Fig. 2AGo). LCA-acetate, which can bind VDR and induce VDR-responsive genes at a micromolar concentration (4, 8), also induced the –211 promoter but not the –128 promoter, again suggesting that a VDR-responsive region resides 5' to the –128 position (Fig. 2BGo). Indeed, either LCA-acetate or 1,25-(OH)2D3 caused a predominantly nuclear localization of the fluorescently labeled [cyan fluorescent protein (CFP)]-VDR in COS-1 (monkey kidney-derived) and NIH3T3 (mouse fibroblast-like) cells, whereas in the absence of the ligand, CFP-VDR was almost uniformly distributed between the cytoplasm and nucleus (data not shown).


Figure 2
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  		Fig. 2. Induction of the Human SULT2A1 Promoter by VDR in Transfected HepG2 Cells

A, 1,25-(OH)2D3-mediated induction of 5'-deleted promoter fragments. B, LCA-acetate-mediated induction of –211 and –128 human SULT2A1 promoters. Luciferase activities were normalized to constant protein amounts. Fold induction was computed relative to the luciferase activity in vehicle (0.01% ethanol)-treated cells. Data are from three independent transfections, each performed in duplicate. Veh, Vehicle.

 
A direct role for VDR in the induced transcription of SULT2A1 is evidenced by chromatin immunoprecipitation (ChIP) assay performed with human HepG2 hepatoma cells (Fig. 3AGo) and with mouse liver samples (Fig. 3BGo). Within 30 min of 1,25-(OH)2D3 treatment, anti-VDR and anti-RXR-{alpha} antibodies precipitated markedly higher amounts of the SULT2A1 gene fragments containing the VDR-response region than that observed for the vehicle-treated cells (Fig. 3AGo). The hormone treatment caused recruitment of the coactivator protein p300 to the same genomic site. The hormone-dependent chromosomal occupancy of VDR, RXR-{alpha}, and p300 was specific because PCR signals were at the background level when the unrelated anti-Cox-2 antibody was used for ChIP (Fig. 3AGo).


Figure 3
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  		Fig. 3. Recruitment of VDR, RXR-{alpha}, and p300 to the Vitamin D-Responsive Region in the SULT2A1 Promoter in Response to Vitamin D Signaling

A, ChIP assay with caco-2 human intestinal cells. B, ChIP assay with samples derived from the mouse liver. Data are representative of the assay results from four (for panel A) and two (for panel B) independent samples. Cox-2, Cyclooxygenase 2; Veh, vehicle.

 
Using liver samples from mice injected with 1,25-(OH)2D3 or EB1089 or vehicle, ChIP assay further showed an agonist-dependent specific recruitment of VDR and RXR-{alpha} to the –168 to –191 region of the mouse Sult2A1 promoter (Fig. 3BGo). In an earlier study we showed that 1,25-(OH)2D3 can induce the –168 to –191 sequence harboring a VDR-binding inverted repeat (IR)0 element (5). The inability of the anti-Cox-2 antibody to pull down the –168 to –191 region demonstrated specificity of the ChIP assay with the tissue samples. These results suggest a direct action of VDR at the vitamin D-responsive site.

Identification of a Vitamin D-Response Element (VDRE) and C/EBP Element within the Vitamin D-Response Region of the Human SULT2A1 Promoter
Deoxyribonuclease I (DNAse I) footprinting of the human SULT2A1 promoter in the presence of recombinant VDR and RXR-{alpha} revealed a binding site for VDR/RXR-{alpha} heterodimer at the –164 to –187 position (Fig. 4AGo). Each receptor alone did not provide the protection. In competition assay using unlabeled double-stranded oligo, the nuclease protection was lost in the presence of the homologous oligo sequence [footprint 2 (FP2)], the VDR-binding IR0 element of the mouse Sult2A1 promoter, and a VDRE from the rat osteocalcin gene. A nuclear factor-{kappa}B (NF-{kappa}B) consensus element did not compete for the footprinted region. However, the mouse liver nuclear extract produced two closely located footprints, one at the –127 to –155 site (FP1) and the other at the –164 to –187 site (FP2) (Fig. 4BGo). The sequences for the two footprint elements (FP1 and FP2) are shown (Fig. 4CGo). FP2 (capable of binding to VDR/RXR-{alpha}) contains an IR2-type imperfect palindromic repeat of AGCTCA, which is similar in sequence to the consensus half-site binding element (A/GGG/TTCA) for all nonsteroid nuclear receptors. Based on sequence homology, we found that the underlined sequence within the FP1 (ATTGCTCAAC) is similar (with only a two-base difference) to the consensus element ATTGCGCAAT for the liver-enriched transcription factor C/EBP (TESS: www.cbil.upenn.edu/tess; TRANSFAC: http://www.cbrc.jp/research/db/TFSEARCH.html).


Figure 4
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  		Fig. 4. DNAse I Footprinting of the Human SULT2A1 Promoter

A, Footprinting produced by recombinant VDR and recombinant RXR-{alpha}. Competition for the footprinted site by various double-stranded oligonucleotides is shown. B, Footprinting produced by mouse liver nuclear extract (NE). The DNA probe was incubated with BSA in the control lanes of panels A and B. C, Nucleotide sequences within FP1 (–155 to –127) and FP2 (–187 to –164). Comp., Competition; cons, consensus.

 
EMSA and antibody supershift confirmed FP2 as a binding site for the heterodimer VDR/RXR-{alpha} and FP1 as the C/EBP-{alpha}/C/EBP-ß binding element. The EMSA complex with the FP2 DNA and mouse liver nuclear extract was competed out by cold homologous oligo DNA and also by VDRE from the rat osteocalcin and mouse Sult2A1 promoter, but not by the nonhomologous NF-{kappa}B consensus sequence (Fig. 5AGo). It appears that proteins other than VDR and RXR-{alpha} also bound to FP2, at least in vitro, because the liver nuclear extract produced multiple EMSA complexes, only some of which (indicated by the arrow; Fig. 5AGo) were competed out by VDRE (osteocalcin) and IR0 (mSult2A1). On the other hand, the homologous IR2 element could compete out all EMSA complexes (Fig. 5AGo). Recombinant VDR and RXR-{alpha} bound specifically to the FP2 element, as evidenced by competition EMSA and antibody supershift, further supporting the conclusion that FP2 is a cognate element for VDR (Fig. 5BGo). The identity of FP1 as the binding site for C/EBP-{alpha} and C/EBP-ß was demonstrated in Fig. 5CGo. The EMSA complex (multiple closely-spaced bands) produced with the FP1 probe and mouse liver nuclear extract was specifically competed out by the unlabeled homologous FP1 sequence and a consensus C/EBP-binding sequence (Fig. 5CGo, lanes 2 and 3) but not by the unlabeled NF-{kappa}B element (lane 4). Antibodies to C/EBP-{alpha} and C/EBP-ß supershifted the EMSA complex (lanes 5 and 6; note asterisk positions); however, the complex was not recognized by the anti-C/EBP-{delta} antibody. Thus, C/EBP-{alpha} and C/EBP-ß can bind to FP1 in vitro.


Figure 5
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  		Fig. 5. EMSA with the FP1 and FP2 Elements

The FP2 probe was incubated with the mouse liver nuclear extract (NE) in panel A and with the recombinant VDR and RXR-{alpha} in panel B. Competition EMSA and antibody supershift results are shown. C, The FP1 probe was incubated with the mouse liver nuclear extract, and the EMSA complex was analyzed for competition by various oligos and for antibody supershift. comp, Competition.

 
An Essential Role of C/EBP-{alpha} in the Functional Response of the SULT2A1 Gene to 1,25(OH)2D3
Disruption of the VDR-binding IR2 motif by point mutations caused a loss of induction of the SULT2A1 promoter by 1,25-(OH)2D3 in transient transfection assays (Fig. 6AGo). The same point mutations also prevented VDR and RXR-{alpha} binding to the IR2 site (data not shown). Intriguingly, point mutations that inactivated the C/EBP binding to the FP1 element also prevented the hormone-mediated induction. Thus, it appears that both FP1 and FP2 elements are involved in the hormonal activation of the SULT2A1 promoter. This possibility was further strengthened by the functional analysis of the FP1 and FP2 elements linked either singly or in combination to a heterologous thymidine kinase (TK) promoter (Fig. 6BGo). 1,25-(OH)2D3 did not induce luciferase expression from the construct containing either element alone as three tandem repeats (3xFP2-TK-Luc) or (3xFP1-TK-Luc) or from the control TK-Luc construct. However, the concurrent presence of FP1 and FP2 elements (3xFP2–3xFP1-TK-Luc) conferred a more than 4-fold induction of luciferase expression in the 1,25-(OH)2D3-treated cells.


Figure 6
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  		Fig. 6. Inducibility of Wild-Type and Mutant Human SULT2A1 Promoters in HepG2 cells by 1,25-(OH)2D3

A, Natural human SULT2A1 promoter with or without inactivating point mutations at the FP1 and FP2 elements. B, The TK promoter linked at the 5'-end to the wild-type and mutagenized FP1 and FP2 elements. Fold induction was computed from the normalized values of three independent duplicate transfections. Veh, Vehicle.

 
Because C/EBP-{alpha} and C/EBP-ß are both expressed in the liver, it was of interest to determine whether a specific C/EBP type is involved in the 1,25-(OH)2D3-mediated regulation. We used NIH3T3 cells to examine the role of each type of C/EBP in the hormonal induction, because in Western blot assay NIH3T3 fibroblast-type cells did not show any significant C/EBP-{alpha} expression, whereas C/EBP-ß, VDR, and RXR-{alpha} were detected readily (see the arrow positions in Fig. 7AGo). When NIH3T3 cells were cotransfected with the vector alone, 1,25-(OH)2D3 failed to induce the natural and heterologous promoter to any significant extent (Fig. 7BGo). In contrast, when these cells were cotransfected with the C/EBP-{alpha} expression plasmid, 1,25-(OH)2D3 induced the natural SULT2A1 promoter (–303 SULT2A-Luc) by approximately 2.5-fold and the heterologous promoter linked to FP1 and FP2 elements (3xFP2–3xFP1-TK-Luc) by about 4-fold in (Fig. 7BGo). Cotransfection of a DNA-binding mutant of C/EBP-{alpha} (C/EBP-{alpha}mt) failed to support the hormonal induction. This DNA-binding mutant was previously characterized for its lack of transactivation function and was designated as C/EBP-{alpha}BR3mt (29). The –128 promoter lacking the VDR-responsive region was not induced by 1,25-(OH)2D3 even upon cotransfection with the wild-type C/EBP-{alpha} plasmid (data not shown). Additionally, the natural SULT2A1 promoter carrying inactivating point mutations at the FP1 element or FP2 element (as described in Fig. 6Go) was not induced by the exogenously expressed C/EBP-{alpha} (data not shown). C/EBP-ß, on the other hand, did not support the vitamin D responsiveness, despite the relatively high endogenous expression of C/EBP-ß in NIH3T3 cells, and even after C/EBP-ß was overexpressed through cotransfection (Fig. 7BGo). Figures 6Go and 7Go together strongly indicate that C/EBP-{alpha} plays an essential role in the SULT2A1 promoter induction by 1,25-(OH)2D3, and the closely related C/EBP-ß is not a functional substitute of C/EBP-{alpha} in this regard.


Figure 7
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  		Fig. 7. Specific Role of C/EBP-{alpha} in the Induction of the Human SULT2A1 Promoter by 1,25-(OH)2D3

A, Expression levels of VDR, RXR-{alpha}, C/EBP-{alpha}, C/EBP-ß, and GAPDH in HepG2, Caco-2, and NIH3T3 cells, as assayed by Western blot. B, Effect of the cotransfected wild-type C/EBP-{alpha} and mutant C/EBP-{alpha} (C/EBP-{alpha}mt) on the promoter induction in NIH 3T3 cells. Histograms indicate average luciferase activities from two independent transfections. Each independent transfection experiment was performed in duplicate wells. C, 1,25-(OH)2D3-mediated induction of the SULT2A1 promoters (–303 and –128) with or without C/EBP-ß cotransfection. Veh, Vehicle.

 
Concurrent Occupancy of C/EBP-{alpha}, VDR, and RXR-{alpha} at the Vitamin D-Responsive Region
A direct involvement of C/EBP-{alpha} at the VDR coactivator complex in the SULT2A1 promoter is evident from kinetic ChiP assay performed on caco-2 cells (Fig. 8Go). Time course analysis showed that recruitment of both VDR and C/EBP-{alpha} to the hormone-responsive region increased markedly within 30 min of 1,25-(OH)2D3 treatment. VDR occupancy diminished by 2 h of the hormone treatment, and C/EBP-{alpha} was mostly gone from the target chromosomal site by 1 h (Fig. 8AGo). The much weaker PCR-amplified signals produced with vehicle-treated cells likely reflect background bands. Because transcription factor occupancy at the regulated site is generally periodic in nature (30), we expect that another cycle of recruitment of VDR and C/EBP-{alpha} to the hormone-responsive site could be observed at later time points. In a second ChIP assay, caco-2 cells were treated with 1,25-(OH)2D3 for 30 min, and recruitment of specific proteins, including C/EBP-ß, was examined (Fig. 8BGo). The absence of C/EBP-ß at the targeted chromosomal site in response to vitamin D treatment is evident, whereas C/EBP-{alpha}, VDR, and RXR-{alpha} can be detected at this site. Therefore, the C/EBP-{alpha} form is specifically involved in the 1,25-(OH)2D3-mediated induction of SULT2A1. These chromosomal events are specific because a nonresponsive upstream sequence in the SULT2A1 promoter (–1613 to –1394) was not immunoprecipitated by the antibodies to VDR, RXR-{alpha}, and C/EBP-{alpha}. Figure 8CGo shows that the p160 coactivators SRC-1 and SRC-2/GR-interacting protein 2 and the global coactivator p300 are present at the hormone-responsive site concurrently with VDR and C/EBP-{alpha}. The corepressor NCoR1 was not recruited. The p160 coactivator recruitment was selective because SRC-3 (amplified in breast cancer 1) was not detected at the regulatory site in 1,25-(OH)2D3-treated cells.


Figure 8
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  		Fig. 8. Concurrent Recruitment of C/EBP-{alpha} and VDR to the Vitamin D-Responsive Region in the Human SULT2A1 Gene

A, Occupancy of VDR and C/EBP-{alpha} to the hormone-responsive region at various time points after 1,25-(OH)2D3 treatment of caco-2 cells. B, ChIP assay on caco-2 cells treated for 30 min with vitamin D or vehicle. Antibodies to VDR, RXR-{alpha}, C/EBP-{alpha}, C/EBP-ß, and COX-2 (an unrelated protein) were used. An upstream region in the SULT2A1 gene devoid of the vitamin D-responsive region was probed as a negative control. C, ChIP assay on caco-2 cells using antibodies to the p160 coactivators SRC-1, SRC-2, and SRC-3; the global coactivator p300; the corepressor NCoR-1; and to VDR and C/EBP-{alpha}. The hormone treatment was for 30 min. Veh, Vehicle; Cox-2, cyclooxygenase 2; ETOH, ethanol.

 
Association between Endogenous VDR and C/EBP-{alpha} in Cells
C/EBP-{alpha} and VDR are in physical association as a complex within the cells, because the antibody against each protein coimmunoprecipitated the other from the cell lysate (Fig. 9AGo). In this assay, lysates from 1,25-(OH)2D3- (or vehicle)-treated caco-2 intestinal cells were immunoprecipitated with the anti-VDR or anti-C/EBP-{alpha} antibody, after which the immunoprecipitates were Western blotted using the reciprocal antibody. As expected, VDR also coimmunoprecipitated with RXR-{alpha}. Similar results were seen with HepG2 cell lysates (data not shown). The interaction between C/EBP-{alpha} and VDR increased markedly in the presence of 1,25-(OH)2D3 (Fig. 9BGo, lanes 1 vs. 2 and 4; lane 6 vs. 7). The interaction is DNA dependent, because coimmunoprecipitation was almost undetectable when the lysates were preincubated with ethidium bromide. Due to its DNA-intercalating property, ethidium bromide is known to disrupt DNA-dependent protein-protein interaction (Fig. 9BGo, lane 3) (31). A DNA fragment containing the VDR-responsive region (FP1+FP2) also enhanced VDR-C/EBP-{alpha} interaction (lane 7 vs. lane 9). Pretreatment of the lysate with calf intestinal phosphatase abolished association of VDR with C/EBP-{alpha} (lanes 5 and 8), indicating that phosphorylation of one or both of these proteins is required to maintain physical interaction between intracellular C/EBP-{alpha} and VDR.


Figure 9
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  		Fig. 9. Coimmunoprecipitation of VDR and C/EBP-{alpha} from caco-2 Cells

A, Antibodies used for IP and Western blot are as indicated. Cells were treated with 1,25-(OH)2D3 for 60 min after which cell lysate was prepared. B, Cell lysates prepared from the hormone-treated cells were incubated with various reagents as indicated and then immunoprecipitated with the anti-C/EBP-{alpha} antibody. Immunoprecipitates were analyzed by Western blot using the anti-VDR antibody. The lysates from vehicle-treated cells were also analyzed (lanes 1 and 6). IP, Immunoprecipitation; WB, Western blot; CIP, calf intestinal phosphatase; EtBr, ethium bromide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Identification of the transcription regulators participating in the basal and induced expression of SULT2A1 is required for a full understanding of the intracellular milieu that affects the SULT2A1-dependent metabolism of substrates originating endogenously (steroids; bile acids) or exogenously (dietary factors; drugs and other xenobiotics). Vitamin D in its active form [1,25-(OH)2D3] triggers increased endogenous SULT2A1 expression in human liver and intestinal cells and in vivo in the mouse liver, and the promoter induction can be recapitulated in transfected cells. In the current study we have examined the role of the liver/intestine-enriched transcription factor C/EBP in the 1,25-(OH)2D3-induced transcriptional response of the human SULT2A1 gene. We show that a composite element containing a VDR/RXR-{alpha} binding site (VDRE) and a C/EBP site, which resides nine bases downstream of the VDRE, mediates the hormonal induction, and this response involves a complete interdependence between VDR and C/EBP-{alpha}. Furthermore, C/EBP-ß does not substitute for C/EBP-{alpha} in this regulation.

The composite nature of the regulatory element and the essential involvement of C/EBP-{alpha} were illuminated by the following results: 1) Inactivating point mutations of the natural SULT2A1 promoter either at the C/EBP site or at the VDRE caused complete loss of the hormone response; additionally, a heterologous promoter was induced by 1,25-(OH)2D3 only in the presence of the composite element, not when either element (VDRE or C/EBP element) was present singly; 2) Cotransfection of C/EBP-{alpha} was absolutely required for the 1,25-(OH)2D3 induction in cells that express a low to nondetectable level of C/EBP-{alpha} (as in NIH 3T3 cells) and a DNA-binding mutant of C/EBP-{alpha} (C/EBP-{alpha}BR3mt) failed to cause the induction; 3) The nonredundancy in the obligatory function of C/EBP-{alpha} is evidenced by the result that C/EBP-ß failed to induce the SULT2A1 promoter in NIH3T3 cells even after cotransfection with a C/EBP-ß expression plasmid. Binding of the VDR/RXR-{alpha} complex at the VDRE and that of C/EBP-{alpha}/ß at the C/EBP site can be demonstrated in vitro based on DNA-protein interaction assays (EMSA and DNAse I footprinting). We ruled out the role of C/EBP-{delta}, another liver-expressed C/EBP member, because the anti-C/EBP-{delta} antibody did not recognize (thus failed to supershift) the EMSA complex produced by incubation of the C/EBP element with the liver nuclear extract.

A direct role of VDR and C/EBP-{alpha} in the induction is evidenced by the result that vitamin D treatment was associated with the recruitment of C/EBP-{alpha}, VDR, RXR-{alpha}, and the p300 coactivator to the response region of the endogenous SULT2A1 gene. Kinetic ChIP assay showed that C/EBP-{alpha}, not C/EBP-ß, was recruited to the responsive region along with VDR and RXR-{alpha} within 30 min of the 1,25-(OH)2D3 treatment. These data further strengthen the conclusion that the functional interaction of C/EBP-{alpha} with the chromatin-associated VDR coactivator complex is essential for the inductive response. Indeed, C/EBP-{alpha} and VDR can associate with each other in vivo because each is able to coimmunoprecipitate the other from cell lysates. The association is DNA dependent, and the composite element enhanced/stabilized the immunoprecipitated complex containing VDR and C/EBP-{alpha}. We speculate that, because of the close proximity of the VDRE and the C/EBP element within the composite response site, physical contact between C/EBP-{alpha} and VDR may create a surface that is optimal for recruiting various coregulators (chromatin remodelers; histone modifiers; mediators). This assembly, in turn, facilitates the formation of the activating macrocomplex that transduces the inductive signal to the general transcription factors and RNA polymerase II. Other examples of VDR target genes that utilize composite elements for induction by 1,25-(OH)2D3 are as follows. In the rat osteocalcin gene, the binding sites for the bone-related master transcription factor Runx2/Cbfa1 flank a VDRE; vitamin D response in human and mouse fibronectin involves cooperation between VDRE and the internally located binding site for the cAMP response element binding transcription factor CREB. It has been suggested that the combined functionality of the DNA-bound VDR/RXR{alpha} and additional nonreceptor transcription factors at a composite response element may facilitate decondensation of the local chromatin structure, which, in turn, would enhance transcriptional response (32).

The specificity of C/EBP-{alpha} over C/EBP-ß, as observed here, is in agreement with many other examples showing the nonredundant functions of these two C/EBP family members, despite their similar binding affinity in vitro for the same DNA sequence and despite the abundant expression of both in the liver. C/EBP-{alpha} has an essential and nonredundant role in the liver expression of several xenobiotic-metabolizing/detoxifying CYP monooxygenases, the bilirubin-detoxifying UDP glucuronosyl transferase and the ammonia-detoxifying enzymes of the ornithine cycle (28). On the other hand, synergism between C/EBP-ß and Sp1 is essential for the developmentally regulated expression of rat Cyp2D5 in the liver, and, in this case, C/EBP-{alpha} is not a functional substitute for C/EBP-ß (33). The VDR coactivator complex in the SULT2A1 promoter selectively recruited SRC-1 and SRC-2 (GR-interacting protein 1/transcriptional intermediary factor 2) but not SRC-3 [p300/cAMPresponse element binding protein-interacting protein (p/CIP)/amplified in breast cancer I (AIBI)] (Fig. 8Go). This selectivity is concordant to the finding that the nuclear receptor regulation of target genes is generally unaffected in SRC-3-inactivated mice. SRC-3 function, however, is more critical for the expression of a subset of genes that have key roles in somatic cell growth (34, 35).

The human SULT2A1 gene provides the first example of a VDRE with an IR2-type configuration of the hexameric core binding sequence (PuGG/TTCA). Direct repeat (DR)3 and DR4 motifs were initially characterized as VDREs in numerous vitamin D target genes (32). It is increasingly common, however, to find alternate repeat motifs such as DR6 (mouse Nas-1 that encodes a sulfate transporter), everted repeat 6 (human CYP3A4; CYP2B6; CYP2C9); everted repeat 9 (mouse c-fos; human calbindin D9K) and IR0 (mouse Sult2A1) as the functional VDR/RXR-{alpha}-binding sequences (5, 32, 36). In theory, the variable architecture and spacer length for VDREs would indicate that both the response element and the DNA-bound receptor can adopt multiple conformations with different interaction surfaces for the various classes of coregulators, thus contributing to gene- and tissue-specific responses to vitamin D signaling. The –211 to –128 region in the human SULT2A1 promoter, harboring the vitamin D-responsive composite element, also mediates induction by the xenobiotic activated nuclear receptors PXR and CAR (our unpublished data). The PXR and CAR signaling appears to utilize both the IR2-type element and a second nuclear receptor-binding site within the –211 to –128 region. Thus, PXR and CAR may compete with the VDR response of the SULT2A1 promoter. Utilization of a common regulatory region by VDR, CAR, and PXR is a recurring theme, because the mouse Sult2A1 promoter is also induced by the activated PXR, CAR, VDR, and FXR via the common IR0 element, and VDR, FXR, and PXR interfere with each other’s ability to transactivate the IR0 element (5). The VDR signaling to drive CYP3A4 induction was also interfered by PXR and CAR due to competitive binding to a common response element (37). Convergence of regulatory signals from different nuclear receptors to a common cis element suggests that the receptor level and conditions that control the intracellular pool of specific ligands (hormones, drugs, nutrients, metabolites) will determine which receptor pathway would be dominant in regulating SULT2A1 expression.

Basal SULT2A1 expression is also expected to be under vitamin D regulation because 1) the 10–50 nM vitamin D concentration that is effective to induce SULT2A1 in human cells and in mice falls within the normal range of the vitamin D level in the adult blood (37); and 2) VDR is expressed in both liver and intestine, the intestinal expression being especially high. Mice inactivated for PXR, or FXR, or combined PXR and FXR are reported to have higher Sult2A1 levels in the liver than wild-type mice (38). This increase is likely due to a compensatory response directed by VDR and possibly by CAR, which also induces mouse Sult2A1 expression (23). Unlike the human promoter that exemplifies a complete interdependence between VDR and C/EBP-{alpha} for the 1,25-(OH)2D3 induction, a 20-bp VDRE with an IR0-type arrangement is sufficient to induce the mouse promoter (5). When formaldehyde-fixed mouse livers were used to isolate nuclei and perform chromatin immunoprecipitation, 1,25-(OH)2D3- and EB1089-dependent occupancy of VDR and RXR-{alpha} at the IR0-containing VDRE in the mouse Sult2A1 promoter was observed (Fig. 3Go). However, whether C/EBP-{alpha} could synergize with VDR to further enhance the mouse gene response to vitamin D signaling was not investigated. Nevertheless, a C/EBP element in the mouse promoter residing 70 bases downstream of the IR0 site is important for the basal activity of the rodent Sult2A1 gene (16). Mice conditionally inactivated for C/EBP-{alpha} in the adult liver are viable (33). Thus, it is now possible to assess whether in vivo C/EBP-{alpha} plays a role (additive/synergistic) in the 1,25-(OH)2D3 induction of mouse Sult2A1.

SULT2A1 induction by LCA via the vitamin D-responsive region (Fig. 3Go) is physiologically significant, especially in the context of bile acid homeostasis, because 1) SULT2A1 is the exclusive enzyme for bile acid sulfation (15); and 2) in humans, sulfonation, rather than CYP-mediated hydroxylation, is the preferred route for the detoxification and disposition of bile acids as water-soluble, readily excreted polar products (14). Bile acids are known to act as carcinogens in human gastrointestinal cancers (39). The SULT2A1 activity is likely to shield enterohepatic tissues and the biliary tract from the bile acid overload that could result from specific metabolic disorders or from an excessive intake of high-fat diets. Experimental evidence for the protective role of SULT2A1 against LCA-induced liver injury comes from the observation that the hepatic SULT2A1 level directly correlates with the extent of liver damage in LCA-injected mice: the higher the SULT2A1 activity, the lower is the extent of injury (38). High urine levels of sulfated bile acids (often as high as 90% of total bile acids), detected in patients suffering from cholestasis, liver cirrhosis, hepatitis, and hepatocellular carcinoma, probably reflected increased bile acid levels arising from disrupted sterol homeostasis (40). Excessive dietary fat may constitute an exogenous source of bile acid overload, because high-fat diets stimulate bile acid secretion and elevate primary and secondary bile acid levels in the fecal excreta (41, 42). The VDR-induced SULT2A1 expression appears to be a defense response to the deleterious endogenous and exogenous substrates of this transferase enzyme. In future studies it will be important to determine which other regulatory factors, in addition to C/EBP-{alpha}, may play key roles in the 1,25-(OH)2D3-stimulated expression of human SULT2A1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Constructs, Mutagenesis, and Transient Transfection
PCR amplification of the human SULT2A1 gene (–1102 to +10) produced promoter fragments with progressively shorter 5'-ends. The amplified products were subcloned into the pCR-BluntII-TOPO vector (Invitrogen, San Diego, CA) and confirmed for sequences. The HindIII-digested promoter inserts were ligated upstream to the cDNA encoding firefly luciferase (pGL3b vector; Promega Corp., Madison, WI). Point mutations were introduced in the –302/+10 promoter at the IR2 (within FP2) and DR4 (within FP1) by PCR-mediated splicing of mutant DNA fragments (right-arm DNA and left-arm DNA). The IR2 mutant was created using the IR2-spanning forward primer 5'-GGAACGCAAGCTTTGATGACCCCT and a reverse primer from pGL3b to create the right-arm mutant DNA; and the IR2-spanning reverse primer 5'-TTAGGGGTCATCAAAGCTTGCGTTCC along with a vector-derived forward primer to generate the left-arm mutant DNA. The underlined bases indicate the sites of mutation. The DR4 mutant was similarly prepared by PCR of the wild-type template with the DR4-spanning forward and reverse primers: 5'-GATAAGTTCATGATTGCTTTACAT; and 5'-GATGTAAAGCAATCATGAACTTAT, respectively, along with the vector-derived forward and reverse primers. A construct with mutations at both IR2 and DR4 (double mutant) was prepared using the DR4 mutant construct as the PCR template and then using the IR2-spanning mutant primers as above. The mutations were confirmed by sequencing.

The heterologous constructs 3x(FP1)-TK-Luc; 3x(FP2)-TK-Luc; and 3x(FP2)-3x(FP1)-TK-Luc were prepared by cloning three tandemly repeated copies of the –155 to –131 (FP1) and –192 to –167 (FP2) sequences into the 5'-end of the TK promoter of the TK-Luc vector using the MluI and BglII cloning sites. Oligonucleotides with suitable restriction sites at 5'- and 3'-ends were custom synthesized (Invitrogen). All constructs were sequence confirmed. The 3A4-PXRE-Luc plasmid contains the homologous xenobiotic enhancer (–7836 to –7208) and human CYP3A4 promoter (–362 to +53) (43).

Cells were maintained in MEM (HepG2 and caco-2 cells) or DMEM (NIH3T3 cells) containing 5% fetal bovine serum. Cells were incubated with 5% charcoal-stripped serum for 3 d before transfection. The cells, seeded overnight (phenol red-free media, 5% charcoal-stripped serum) in 24-well flasks at approximately 100,000 cells per well, were transfected with various DNA constructs using Fugene (Roche-Boehringer Mannheim Biochemicals, Indianapolis, IN). Each well contained the reporter construct at 400 ng, human VDR plasmid at 50 ng, and human RXR-{alpha} at 10 ng. For experiments with NIH3T3 cells, wild-type C/EBP-{alpha} or the C/EBP-{alpha}BR3 mutant was used at 50 ng per well. The BR3 mutant has point mutations within the DNA-binding domain of C/EBP-{alpha} and thus has lost DNA-binding ability (29). After overnight DNA transfer, cells were incubated with 1,25-(OH)2D3 (at 50 nM), LCA-acetate (at 10 µM), or vehicle (<0.01% ethanol) for 24 h, and the cell extracts were assayed for firefly luciferase activity. Reporter expression was normalized to constant protein amounts. Results were computed from three independent transfection experiments, each conducted in duplicate.

ChIP Assay
ChIP was performed on nuclear lysates from cells grown in culture or from mouse livers. Cells were grown to 90% confluence in 10-cm tissue culture dishes by culturing for 3 d in phenol red-free DMEM supplemented with 5% charcoal-dextran-stripped fetal bovine serum. After changing to a fresh charcoal-stripped serum-containing medium, the cells were treated with 1,25-(OH)2D3 (at 20 nM) for various times. At each time point the cells were treated with 1% formaldehyde for 10 min at 37C (gentle shaking), and protein-DNA cross-linking was stopped by incubation with glycine (125 mM) at room temperature for 5 min. Afterward, the cells were washed twice with ice-chilled PBS (pH 7.5) and lysed in 0.5 ml of a lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)]. Cell lysates were sonicated with six separate bursts on ice (each burst lasting 25 sec; 2-min resting interval between each burst). The sonicated samples were centrifuged (4 C, 10 min, 13,000 rpm). The solubilized chromatin fragments in the supernates were diluted 10-fold with a dilution buffer (0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA; 16.7 mM Tris-HCl, pH 8.0; 167 mM NaCl) before immunoprecipitation.

For ChIP with mouse liver samples, nuclei were prepared from livers as described (44). Livers (~2 g) were treated with 1% formaldehyde (gentle shaking for 10 min at room temperature) and incubated further with 125 mM glycine solution (shaking for 10 min at room temperature). The fixed tissue was diluted with 2 volumes of lysis buffer (5 mM HEPES, pH 8.0; 85 mM KCl; 0.5% Nonidet P-40; and a protease inhibitor cocktail; Sigma Chemical Co., St. Louis, MO), homogenized for 20 sec on ice, and incubated further on ice for 15 min. The homogenate was centrifuged (3500 x g, 5 min), and the nuclei were lysed with a buffer (10 mM EDTA; 1% SDS; 50 mM Tris-HCl, pH 8.1) at 1:1 (wt/vol) relative to the initial tissue weight. The lysate was incubated (ice, 10 min), spun quickly (4 C), sonicated as above, and processed to isolate chromatin fragments.

For ChIP, samples were treated with an antibody sample at 500 ng per reaction (4C, overnight incubation under rotation). The cross-linked protein-DNA complexes in the immunoprecipitate were bound to protein A-agarose (40 µl solution for each precipitated sample; Upstate Biotechnology, Inc., Lake Placid, NY) and washed sequentially with low-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl), high-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0; 500 mM NaCl) and LiCl buffer (250 mM LiCl; 1% Nonidet P-40; 1% deoxycholic acid; 1 mM EDTA; and 10 mM Tris-HCl, pH 8.0). Final wash was with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (twice), and the immune-precipitates were eluted (room temperature) with a freshly prepared elution buffer (1% SDS and 0.1 M sodium bicarbonate). The eluate at 200 mM NaCl was heated (65 C, 4 h) to reverse cross-linking. Genomic DNAs were isolated from proteinase K-treated (45 C for 1 h) samples and purified (Zymo Research Corp., Orange, CA) and were PCR amplified at the target region.

The VDR-responsive region in the human SULT2A1 gene was amplified with the forward primer (5'-CTTGCAGTTCACTCTCAGGA at –209); reverse primer (5'-CACCGCTGGAGGCTGTGGAC at +22). An upstream sequence lacking a VDR-responsive region was amplified with 5'-CCTCGGCCTCCCAAAGTGCT (forward primer at –1613); 5'-AAAGCTGAATAGAAGTCTAC- (reverse primer at –1394). The VDR-responsive region in the mouse Sult2A1 promoter was amplified with 5'-GCATTTCTATGTCCTATTAC (forward at –231); 5'-GATATGATATGGCAGGAAAAGGT (reverse at + 81).

Hormone Treatment, RNA Isolation, and Northern Blot
Mice were injected ip with 1,25-(OH)2D3 or EB1089 for 3 d at 1.5 µg hormone/mouse/d. Control mice were injected with propylene glycol (100 µl at 0.05M in PBS). All animal experiments conformed to the approved institutional animal care protocol. Total RNAs isolated by Trizol (Invitrogen) were processed for Northern blot. The RNAs transferred onto a nylon membrane (Ambion, Inc., Austin, TX) were hybridized to 32P-labeled cDNA probes for mouse Sult2A1, or Cyp3A11, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The radiolabeled probes were prepared by random priming (Rediprime II kit; Amersham Pharmacia Biotech, Arlington Heights, IL). The N-terminal end of mouse Sult2A1 was used as the cDNA probe to ensure the absence of cross-reactivity with any other members of the SULT family (45). RT-PCR of the mouse liver total RNAs produced the cDNA fragments for Sult2A1 and Cyp3A11, and PCR products were sequence confirmed. RT-PCR primers are as follows. Mouse Sult2A1: 5'-GAAGGCATACCTTTTCCTGCCAT (forward primer at +51) and 5'-GTAACCAGACACAAGAATATCTCT (reverse primer at +419). Cyp3A11: 5'-TCATCCCAGCAAAAATAAA (forward primer at +612); 5'-TCATCC CAGCAAAAATAAA (reverse primer at +1003). The radiolabeled mouse GAPDH probe was prepared from the pTRI-GAPDH plasmid (Ambion). After autoradiography Northern blot filters for Sult2A1 and Cyp3A11 were stripped off the radiolabeled probe and rehybridized with the 32P-labeled GAPDH cDNA. The Northern signals were quantified using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

DNAse I Footprinting and EMSA
A radiolabeled coding strand probe for DNAse I footprinting was generated by PCR of the human SULT2A1 promoter using a 32P-end-labeled forward primer (at –302) and an unlabeled reverse primer from within the vector (pGL3b). The conditions for footprinting assay and mouse liver nuclear extract preparation were described elsewhere (12). Recombinant human VDR (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) and human RXR-{alpha} (as a glutathione-S-transferase fusion) were used in some experiments.

For EMSA, the liver nuclear extracts or recombinant proteins were preincubated (5 min at room temperature) with polydeoxyinosinic deoxycytidylic acid (0.1–0.3 µg/µl) in a DNA-binding buffer (10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM EDTA; 5% glycerol (vol/vol); 1 mM dithiothreitol) after which the oligo probe was added and incubation continued for 30 min (room temperature). The DNA-bound proteins were analyzed on a 5% native polyacrylamide gel. In competition EMSA and antibody supershift, the unlabeled competitor or the test antibody was added at preincubation. Antibodies to RXR-{alpha} ({Delta}N 197), VDR (D-6), and C/EBP-{alpha}, -ß, and -{delta} were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Coimmunoprecipitation and Western Blot
The caco-2 human intestinal cells express VDR and C/EBP-{alpha} endogenously at relatively high levels; thus these cells were used for coimmunoprecipitation. Cells grown to confluence in 10-cm dishes were treated with 1{alpha},25-(OH)2D3 (20 nM) or vehicle (ethanol, <0.01%) for different time points and subsequently lysed by incubating for 30 min at 4 C in 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin. The cell lysate was cleared by spinning at 12,000 x g for 10 min. Samples (500–700 µg protein) were incubated (overnight at 4C under rotation) with anti-C/EBP{alpha} or anti-VDR antibodies, and the immunoprecipitates were pulled down by protein A-Sepharose beads and washed with cold lysis buffer (four times) and once with cold 20 mM Tris-HCl buffer (pH 7.5). The immunoprecipitates were boiled with Laemmli’s buffer and analyzed by Western blot using an enhanced chemi-luminescence signal detection system (Pierce Chemical Co., Rockford, IL).


   ACKNOWLEDGMENTS
 
We thank Dr. Lise Binderup (LEO Pharma, Ballerup, Denmark) for EB1089; Dr. Robert Christy (San Antonio, TX) for the C/EBP-{alpha} and C/EBP-ß expression plasmids; Dr. Rommel Tirona (Vanderbilt University, Nashville, TN) for the 3A4-PXRE-Luc plasmid; and Dr. Ronald Evans (The Salk Institute, La Jolla, CA) for the RXR-{alpha} plasmid.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant AG-10486 (to B.C.); a grant from Philip Morris USA (to B.C.); and a Merit Review grant (to B.C.) and Career Scientist Award (to B.C.) from the Department of Veterans Affairs.

Authors’ Disclosure Summary: All authors have nothing to declare.

First Published Online December 15, 2005

Abbreviations: CAR, Constitutive androstane receptor; C/EBP, CAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; CYP, cytochrome P450; DNAse I, deoxyribonuclease I; DR, direct repeat; FP, footprint; FXR, farnesoid X receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; IR, inverted repeat; LCA, lithocholic acid; NF-{kappa}B, nuclear factor-{kappa}B; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; PXR, pregnane X receptor; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; TK, thymidine kinase; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication October 25, 2005. Accepted for publication December 9, 2005.


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 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   VDR  |  RXRα
Coregulators:   p300  |  SRC-1  |  GRIP1  |  AIB1  |  NCOR
Ligands:   Calcitriol



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