The alternatively spliced murine pregnane X receptor isoform, mPXRΔ171–211 exhibits a repressive action

The International Journal of Biochemistry & Cell Biology 42 (2010) 672–682

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The alternatively spliced murine pregnane X receptor isoform, mPXR171–211 exhibits a repressive action Marko Matic a , Anthony P. Corradin a,b , Maria Tsoli a , Stephen J. Clarke a , Patsie Polly a,b,1 , Graham R. Robertson a,∗,1 a b

Cancer Pharmacology Unit, ANZAC Research Institute, Hospital Road, Concord RG Hospital, NSW 2139, Australia Department of Pathology, University of New South Wales, NSW 2052, Australia

a r t i c l e

i n f o

Article history: Received 3 September 2009 Received in revised form 5 December 2009 Accepted 4 January 2010 Available online 11 January 2010 Keywords: Pregnane X receptor Variant isoform Repressive function CYP3A4 MDR

a b s t r a c t The orphan nuclear receptor pregnane X receptor regulates enzymes and transport proteins involved in the detoxification and clearance of numerous endobiotic and xenobiotic compounds, including pharmaceutical agents. Multiple alternatively spliced pregnane X receptor isoforms have been identified which are significantly expressed in humans and mice (up to 30% of the total pregnane X receptor transcript), however, little is known about their biological action. We explored functional differences between the major mouse pregnane X receptor isoforms mPXR431 and mPXR171–211 that lacks 41 amino acids adjacent to the ligand-binding pocket. Transient transfection assays showed that mPXR171–211 reduced the basal transcription of cytochrome P450 3A4 and the drug transporter P-glycoprotein/Multi Drug Resistance Protein 1 and directly repressed the regulatory effects of mPXR431 on these genes. Replacement of the mPXR171–211 DNA-binding domain with that of GAL4 showed mPXR171–211 retained its repressive role independent of binding to PXR responsive elements located within the cytochrome P450 3A4 and Multi Drug Resistance Protein 1 regulatory regions. Use of the histone deacetylase inhibitor, trichostatin A, demonstrated that the repressive function of mPXR171–211 acts independently of histone acetylation state. Protein interaction assays revealed mPXR171–211 and mPXR431 differentially bind the obligatory heterodimer partner retinoid X receptor. Furthermore, mPXR431 and mPXR171–211 proteins could heterodimerize. These studies demonstrate that the variant mouse PXR isoform, mPXR171–211 , has a distinct repressive function from mPXR431 in regulating genes encoding important drug metabolizing enzymes and transport proteins. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Pregnane X receptor (PXR; NR1I2 – also known as the steroid and xenobiotic (Blumberg et al., 1998) or pregnane activated receptor (Bertilsson et al., 1998)) is a member of the nuclear receptor (NR) family of ligand-activated transcription factors. Nuclear receptors regulate a wide range of physiological processes including devel-

Abbreviations: PXR, pregnane X receptor; CYP, cytochrome P450; MDR1, Multi Drug Resistance Protein 1; P-gp, P-glycoprotein; TSA, trichostatin A; RXR, retinoid X receptor; NR, nuclear receptors; h, human; m, mouse; mRNA, messenger ribonucleic acid; mP1, mPXR431 ; mP2, mPXR171–211 ; CAR, constitutive androstane receptor; DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; GST, glutathioneS-transferase; VDR, vitamin D receptor; PR, progesterone receptor. ∗ Corresponding author. Tel.: +61 2 9767 7109; fax: +61 2 9767 8069. E-mail addresses: [email protected] (M. Matic), [email protected] (A.P. Corradin), [email protected] (M. Tsoli), [email protected] (S.J. Clarke), [email protected] (P. Polly), [email protected] (G.R. Robertson). 1 Joint senior author. 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.001

opment, cell differentiation and organ function. Classical nuclear receptors such as steroid hormone receptors have high ligand specificity and sensitivity, usually carrying out transcriptional regulation as a homodimer. However, PXR is a member of the orphan nuclear receptor sub-family for which a cognate physiological ligand has not yet been identified. Orphan receptors such as PXR are capable of binding a wide range of ligands with low specificity. In addition, rather than acting as homodimers, orphan nuclear receptors usually carry out their transcriptional regulation as heterodimers with the retinoid X receptor (RXR). PXR plays an important role in coordinating clearance of xenobiotic and endobiotic compounds within the body. It regulates genes encoding enzymes involved in biotransformation as well as transporter proteins for uptake and efflux of foreign substances, including pharmaceutical compounds (Gibson et al., 2006; Tirona and Kim, 2005). One of the best characterized PXR targets is the cytochrome P450 3A4 (CYP3A4) gene, which encodes the most abundant cytochrome P450 enzyme in the human liver (Ingelman-Sundberg, 2004). As the CYP3A4 enzyme is involved in the metabolism of up to 60% of all currently marketed therapeutic

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agents (Hustert et al., 2001; Liu et al., 2008), it is quantitatively and qualitatively one of the most important enzymes in human drug metabolism. Many of the compounds known to induce CYP3A4 also activate PXR, consequently up-regulating drug clearance pathways, thereby increasing the rate of drug elimination. This forms the basis of many clinically important PXR-mediated drug interactions involving altered pharmacokinetics of therapeutic compounds. An additional level of complexity to nuclear receptor action occurs through existence of multiple isoforms (Keightley, 1998). These can arise through alternative mRNA splicing events, transcriptional initiation sites or via different genomic loci. Such isoforms can have tissue-specific expression patterns, differential sub-cellular localization and interactions with proteins and/or DNA response elements. These properties can result in loss, gain or alteration of function. Full-length PXR sequences have been characterized in mouse (Kliewer et al., 1998), human (Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998), rabbit and rat (Jones et al., 2000; Zhang et al., 1999). Partial PXR sequences encompassing the ligand-binding domain (LBD) have been cloned from pig, dog, rhesus monkey, zebra and fugu fish (Maglich et al., 2003; Moore et al., 2002). Human PXR (hPXR) can exist as 15 isoforms, while two mouse PXR (mPXR) isoforms were described in the initial discovery of PXR (Kliewer et al., 1998) (Table 1). The expression level and composition of human PXR isoforms can vary dramatically between individuals (He et al., 2006; Lamba et al., 2004). Additional rabbit and rat PXR liver transcripts have been identified, however these have not been extensively studied (Jones et al., 2000; Zhang et al., 1999). The majority of research has focused on full-length human PXR (i.e. hPXR434 ), whose dominant expression and susceptibility to drug activation have established its potential to impact on clinical outcomes via inductive drug interactions. Beyond hPXR434 , significantly expressed hPXR isoforms include splice variants hPXR174–210 and hPXR174–214 [designated PXR.2 and PXR.3 (Lamba et al., 2004)] and another splice variant which generates a premature stop codon at residue 196 [designated SV3 (He et al., 2006)]. The combined expression of these isoforms can contribute up to 28% of the total hPXR mRNA transcripts (Lamba et al., 2004). hPXR174–210 and hPXR174–214 are virtually the same, possessing a deletion of 111 and 123 nucleotides, respectively in exon 5 of the hPXR gene. The precise 41 amino acids (aa) exclusion found in hPXR174–214 is conserved in the only mouse PXR isoform identified to date, mPXR171–211 . Limited functional characterization of the mouse PXR isoforms comparing full-length mPXR431 and mPXR171–211 (herein designated mP1 and mP2, respectively) revealed that they are both capable of binding the same responsive elements within the CYP3A gene. However, mP2 has a reduced ligand activation profile compared to mP1 (Kliewer et al., 1998). Elucidation of the hPXR434 LBD crystal structure revealed a flexible loop region at the ligand entry to the ligand-binding pocket (Watkins et al., 2001) which overlaps the amino acids corresponding to the region deleted in the variant isoforms mP2, hPXR174–210 and hPXR174–214 (Fig. 1). The lack of this flexible loop could explain the restricted activation profile of mP2 relative to the promiscuous mP1. Interestingly, alignment of PXR and its isoforms, with other nuclear receptors reveals the missing region of mP2, hPXR174–210 and hPXR174–214 is also absent in other nuclear receptors such as constitutive androstane receptor (CAR), farnesoid X receptor and liver X receptor (Ekins et al., 2002; Lamba et al., 2004; Matic et al., 2007) which play important roles in a range of metabolic processes. This, together with evidence of tissue-specific expression patterns of isoforms in a wide variety of organs (including liver, stomach, adrenal gland, bone marrow and brain) (Fukuen et al., 2002; Kliewer et al., 1998; Lamba et al.,

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2004) suggests these PXR isoforms may possess important biological functions. In this study, we demonstrate that the variant mouse isoform mP2 is functionally distinct from full-length mP1. mP2 by itself reduces the basal transcription of CYP3A4 and MDR1 and directly represses the regulatory effects of mP1 on these genes. 2. Materials and methods 2.1. Plasmid constructs for in vitro translation and mammalian expression A modified pSG5 vector backbone, pSG5EX , was generated by ligating annealed pSG5EX (F) 5 -AATTGCTCGAGAGCGGCCGCAGAATTCAA-3 and pSG5EX (R) 5 -GATCTTGAATTCTGCGGCCGCTCTCGAGC-3 oligonucleotides into the EcoRI and BglII sites of the pSG5 vector (Stratagene, CA, U.S.A.). This abolished the pSG5 EcoRI and BamHI and introduced XhoI, NotI and EcoRI sites. The pSG5EX mPXR431 and pSG5EX mPXR171–211 constructs were generated by PCR amplification of mPXR431 and mPXR171–211 cDNA fragments from cDNA derived from total mouse liver RNA (Nakhel, S. and Robertson, G.R., unpublished). The following primers were used: (F) 5 -ATCTCGAGCGCCACCATGAGACCTGAGGAGAGCTGG-3 XhoI restriction site, Kozac translation sequence, start codon and (R) 5 -GAGAATTCTCAGCCATCTGTGCTGCTAAATAACTCTTGC-3 EcoRI restriction site, stop codon). These primers incorporated an XhoI restriction site and a consensus Kozac translation sequence immediately upstream of the mPXR start codon, in addition to an EcoRI restriction site immediately downstream of the stop codon. The amplicon was ligated into the pGEM-T vector (Promega, New South Wales, Australia) then digested using EcoRI and XhoI followed by sub-cloning into the EcoRI and XhoI sites of pSG5EX to generate pSG5EX mPXR431 and pSG5EX mPXR171–211 . pSG5–GAL4–PXR.2LBD encoding mPXR171–211 LBD fused to a GAL4 was a kind gift from Prof. Steve Kliewer (University of Texas Southwestern Medical Centre, Dallas, U.S.A. (Kliewer et al., 1998)). Prof. Carsten Carlberg (University of Luxemburg, Luxembourg) provided the human RXR␣ expression construct pSG5hRXR␣. 2.2. Plasmid constructs for bacterial expression The bacterial expression constructs pGEX-mPXR431 and pGEXmPXR171–211 were generated by PCR amplification of cDNA encoding amino acids 1–431 of mPXR431 or 1–390 of mPXR171–211 followed by insertion into EcoRI (GAATTC) and XhoI (CTCGAGC) sites of a pGEX-4T vector. The PCR primer sequences corresponding to nucleotides 1–21 and 1275–1296 of the mPXR coding region were: (F) 5 -TCAGAATTCATGAGACCTGAGGAGAGCTGG-3 ; (R) 5 CATCTCGAGTCAGCCATCTGTGCTGCTAAA-3 . 2.3. Reporter gene plasmid constructs The p3A4-13000, p3A4-362(7836/7208 ins) reporter gene constructs containing DNA response elements from the CYP3A4 upstream promoter were a kind gift from Prof. Chris Liddle (Westmead Millennium Institute, Westmead, Australia (Goodwin et al., 1999)). The p-7975(7012–1804) reporter gene construct containing DNA response elements from the MDR1 upstream regulatory region was kindly provided by Prof. Oliver Burk (Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany (Geick et al., 2001)). All reporter constructs consist of segments of the respective gene’s upstream regulatory region directly linked to the luciferase gene contained within the pGL3 basic vector (Promega, Madison, WI).

674 Table 1 Schematic representation of human and mouse PXR isoforms. The exons are numbered and depicted as open boxes except exon 5 which is diagonally shaded. Arrows indicate alternate start codons in each transcript. Termination codons of open reading frames are depicted as asterisks. Intronic sequence insertions are shown as single lines between exon boxes shaded in black. DBD, DNA-binding domain; LBD–ligand-binding domain. a Bertilsson et al. (1998), b Blumberg et al. (1998), c Dotzlaw et al. (1999), d Fukuen et al. (2002), e Kliewer et al. (1998), f Lamba et al. (2004), and g Lehmann et al. (1998).

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Fig. 1. Protein sequence comparisons of the major PXR isoforms in context of the nuclear receptor NR1I sub-family. Protein sequence alignment of the nuclear receptors VDR, CAR and PXR including the major human and mouse PXR isoforms at the differentially spliced exon 5 domain of the PXR mRNA. The major human and mouse PXR isoforms are schematically represented, with the corresponding aligned regions encompassing the differentially spliced domain shaded in black. The sequence alignment highlights the similarity of the long hPXR and mPXR isoforms with VDR, while the shorter PXR variants share a similar regional deletion present in CAR.

2.4. Cell transfections and luciferase reporter gene assays Human hepatocellular carcinoma (HepG2), African green monkey kidney (Cos7) and human intestinal adenocarcinoma (LS180) cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (penicillin 50 units/ml, streptomycin 50 ␮g/ml). Cells were typically seeded into 24 well plates (2 × 104 cells/cm2 ) (Becton Dickinson, New South Wales, Australia) and maintained at 37 ◦ C and 5% CO2 for 24–48 h prior to transfection. Transfections were performed by forming liposomes with FuGene (Roche Applied Science, New South Wales, Australia), 300 ng of luciferase reporter, 100 ng of the pCMV␤ reference construct and 100 ng of PXR containing expression constructs (unless otherwise stated) for 15 min at room temperature in a total volume of 1 ml DMEM. Transfection was carried out by addition of liposomes to the cells for 24 h followed by incubation with fresh medium for an additional 48 h to examine basal PXR-mediated transcriptional effects. Transfected cells were tested for PXR activation or inhibition of transcription by treatment with vehicle, ligand, or ligand and trichostatin A (TSA) for an additional 24 h. The selection of TSA concentration (20 nM) was based on our previous experience with TSA (Polly et al., 2000), and that of other studies (Li and McDonnell, 2002; Zhong et al., 2002). Cells were disrupted with reporter lysis buffer (Promega, New South Wales, Australia) and luciferase reporter activity was quantified using a reporter assay system (Promega, New South Wales, Australia) using a Victor III multi-plate reader (PerkinElmer, New South Wales, Australia). The luciferase activities were normalized with respect to ␤-galactosidase activity (Foster et al., 1988).

were grown at 37 ◦ C with constant shaking until an OD600 reading of 0.3–0.4 was attained. Protein expression was induced by the addition of isopropyl-␤-d-thio-galactopyranoside (IPTG; 1 mM), with incubation for an additional 4 h at 30 ◦ C. Bacterial cultures were centrifuged and cell pellets frozen at −80 ◦ C. 2.6. GST fusion protein purification Ice cold STE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8), containing protease inhibitors, was added to each bacterial pellet. Lysozyme was added to the suspension and incubated for 30 min on ice. Following addition of 0.2 ml DTT (1 M) and 4.5 ml 10% Triton-X 100, cell lysates were sonicated and centrifuged at 12,000 rpm for 15 min at 4 ◦ C. Glutathione sepharose 4B beads were washed, added to the cleared supernatant and mixed for 1 h at 4 ◦ C. The slurry was centrifuged for 5 min, 500 rpm at 4 ◦ C, and then washed with PBS (5×), 1.5 M NaCl in PBS (1×), and finally in PBS (2×). Purity of proteins was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie brilliant blue staining. Following purification of GST fusion proteins, Western blot analysis was carried out with an anti-GST antibody. 2.7. In vitro protein translation In vitro translated proteins mPXR434 , mPXR171–211 and hRXR␣ were generated from pSG5EX mPXR434 , pSG5EX mPXR171–211 and pSG5RXR␣ by using TNT® coupled transcription–translation reactions (Promega, New South Wales, Australia). Protein products were routinely visualized and quantified by [35 S]-methionine incorporation.

2.5. Expression of glutathione-S-transferase (GST) fusion proteins 2.8. GST pull-down assays Three GST constructs (pGEX-0, pGEX-mPXR431 , pGEXmPXR171–211 ) were transformed into chemically-competent BL-21LysSGold bacteria (Stratagene, California, U.S.A.). Cultures

Bacterial over-expression of GSThRXR␣, GSTmPXR431 and GSTmPXR171–211 , fusion proteins was performed by induction

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with IPTG (1.25 mM) for 3 h at 37 ◦ C in the JM109 E. coli strain. Fusion proteins were checked for equal loading by Coomassie brilliant blue staining. GST pull-down assays were performed by incubation of GSThRXR␣ with either [35 S]-mPXR434 or [35 S]mPXR171–211. A 50% GSThRXR␣-sepharose bead slurry in PPI buffer (20 mM HEPES, pH 7.9; 200 mM KCl; 1 mM EDTA, 4 mM MgCl2 , 1 mM dithiothreitol, 0.1% Nonidet P-40 and 10% glycerol). GST fusion-sepharose slurries were pre-blocked in PPI buffer containing bovine serum albumin (1 ␮g/␮l) prior to use in pull-down assays. [35 S]-mPXR434 or [35 S]-mPXR171–211 labeled proteins that were not bound to GSThRXR␣ were washed away with PPI buffer. The quantity of in vitro translated [35 S]-mPXR434 or [35 S]-mPXR171–211 used (10% of input) and bound to GSThRXR␣ was detected by electrophoresis through a 10% SDS-PAGE, and following drying, densitometry analysis was performed on an autoradiographed gel image using ImageJ software (Abramoff et al., 2004). 2.9. Electrophoretic mobility shift assays DNA–protein complex reactions were typically performed in a total volume of 20 ␮l binding buffer (50 mM HEPES, pH 7.8; 10 mM KCl; 5% glycerol; 1 mM polyDI-DC), approximately 5 ng of each in vitro translated mPXR434 , mPXR171–211 and hRXR␣ and 1 ng dNR1 or prPXRE 32 P-labelled double stranded oligoncleotides described elsewhere (Goodwin et al., 1999). Complexes were incubated for 20 min at room temperature then resolved through 6% (w/v) nondenaturing polyacrylamide gels in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA (pH 8.3)) for 150 min, 200 V at 4 ◦ C. Gels were dried at 80 ◦ C for 90 min and visualized by autoradiography using Kodak BIOMAX MR Film (Sigma, New South Wales, Australia). Densitometry analysis on resolved bands was performed using ImageJ software (Abramoff et al., 2004). 2.10. In vivo mPXR isoform expression 10–14-week old male FVB strain mice were examined for PXR isoform expression in liver, kidney and intestine (duodenum and jejunum). Animal studies were conducted in accordance with the guidelines of the Australian Council on Animal Care. Animals were kept in a temperature-controlled facility with 12-h light/dark cycles and were fed a standard rodent chow diet with water ad libitum. Total RNA was extracted from snap frozen tissue using Trizol (Invitrogen, Victoria, Australia) and the Pro200 homogenizer (Daintree Scientific, Tasmania, Australia) and the concentration and purity of RNA were determined from the absorbance at 230, 260 and 280 nm using the Biophotometer (Eppendorf, New South Wales, Australia). cDNA was synthesized from total RNA (2 ␮g) using SuperscriptIII according to the manufacturer’s instructions (Invitrogen, Victoria, Australia). PCR analysis was performed using BIO-X-ACT Short Mix (Bioline, New South Wales, Australia) according to the manufacturer’s instructions with the following (F) 5 -GAAAAGATTGAGGCTCCACC-3 , and (R) 5 TTTGGCGAAGTTGATGACGC-3 primers, which flank exon 5 of mouse PXR with thermocycling regime; 95 ◦ C, 5 min; [95 ◦ C, 20 s; 63.7 ◦ C, 30 s; 72 ◦ C, 30 s] for 35 cycles; 72 ◦ C, 5 min. cDNA (2 ␮g) was used for liver and intestine samples, and twice that for kidney

samples. pSG5EXmPXR431 and pSG5EXmPXR171–211 (0.1 ng) were used as positive controls and water as a negative. 2.11. Data analysis and statistics Quantitative data were expressed as mean ± standard error of the mean (SEM). Statistical analyzes among groups were performed using the Student’s t-test. Significance was set at p ≤ 0.05. 3. Results 3.1. Expression of the variant PXR isoform mP2 (mPXR171–211 ) in mouse tissues The relative abundance of mP1 and mP2 transcripts was examined in total cDNA of individual mouse liver, intestine and kidney samples. Isoform-specific amplicons were generated using PCR primers spanning the differentially spliced mPXR exon 5, which is absent in mP2. Agarose gel electrophoresis of the PCR products revealed two bands corresponding to the expected sizes of mP1 (405 base pairs) and mP2 (282 base pairs) (Fig. 2). While the major product representing full-length mPXR is more abundant, the short isoform is still expressed at significant levels and is therefore not a rare transcript. 3.2. mP2 represses the basal transcription levels of target genes The transcriptional activity of the two mouse PXR isoforms was analyzed using chimeric CYP3A4-Luc and MDR1-Luc reporter constructs in transient transfection assays. Human hepatocellular carcinoma (HepG2), human intestinal adenocarcinoma (LS180) and African green monkey kidney (Cos7) cells were chosen as representative cell lines derived from organs known to express PXR transcripts (Fig. 2), and as previously reported (Kliewer et al., 1998). Assessment of mP1 and mP2 transcriptional activity in HepG2 cells on the p3A4-13000 reporter, containing the full-length CYP3A4 promoter and enhancer (Xenobiotic Responsive Enhancer Module [XREM], −13 kb) revealed a 10-fold mP1 and a 0.4-fold mP2 transcriptional readout in reference to vector control (Fig. 3A). Similar results were observed on the p3A4-362(7836/7208 ins) reporter consisting of the XREM region positioned immediately adjacent to the proximal CYP3A4 promoter with the intervening DNA sequences of the CYP3A4 upstream region omitted. mP1 readout was 30-fold and mP2 was 0.5-fold that of vector control. Transcriptional activity of the two isoforms was further examined on another well-defined PXR target gene, the P-glycoprotein/Multi Drug Resistance Protein 1 (P-gp/ MDR1) using the p-7975(7012–1804)-Luc reporter (Geick et al., 2001). On this reporter, a trend indicating mP1 increased the reporter readout (compared to vector), while mP2 repressed it to 0.3-fold that of vector control was noted (Fig. 3A). In addition to HepG2 cell lines, similar mP2-mediated repressive effects were observed in human intestinal adenocarcinoma (LS180) and African green monkey kidney (Cos7) cells (data not shown). To demonstrate that the repressive effect was mP2 specific, the transcriptional activity of the p3A4-13000 construct was quantified against titrated transfected amounts of mP2 in Cos7 cells.

Fig. 2. mP2 (mPXR171–211 ) represents a significant component of the total mPXR transcript. Semi-quantitative PCR of mPXR431 (mP1) and mP2 gene transcripts encompassing the differentially spliced exon 5 region was performed on individual mouse liver, intestine and kidney cDNAs. PCR products were resolved on a 2% agarose gel.

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Fig. 4. The variant PXR isoform mP2 represses mP1-mediated basal transcription on CYP3A4 gene. Transcriptional regulation of transiently co-transfected mP1, mP2 and V (indicated in ␮g) was evaluated on the p3A4-13000 in Cos7 cells. Transfection quantities are indicated in ␮g. Data are shown as fold basal transcriptional activity and represent the mean of three independent experiments performed in triplicate.

3.4. mP2 interferes with mP1 induction of the CYP3A4 gene in response to ligand activation

Fig. 3. mP2 represses the basal transcription levels of CYP3A4 and MDR1 genes. (A) Transcriptional regulation by transiently transfected mouse PXR isoforms on co-transfected CYP3A4 and MDR1 reporter constructs. Equal quantities (0.1 ␮g) of mP1, mP2 or vector control (V) were individually co-transfected with either of the two CYP3A4-Luc reporter constructs [p3A4-13000 or p3A4-362 (7836/7208 ins)] or the MDR1-Luc reporter construct [p-7975(7012–1804)] in HepG2 cells. (B) Transcriptional regulation of a range of mP2 quantities (0.01–0.6 ␮g) was evaluated on the p3A4-13000 reporter in Cos7 cells. The reporter readout was monitored based on luciferase production. Data are presented as fold basal expression as indicated by vector (V) readout, and represent the mean of three independent experiments. Error bars indicate standard error of the mean (SEM). Statistical analysis was performed in reference to V through application of the Student’s t-test. *p ≤ 0.05, ***p ≤ 0.0001.

Reporter readout was significantly repressed at 50 and 100 ng of co-transfected mP2. While the degree of repression appears to be reduced at 600 ng, it is still present at this high level of total input DNA. Importantly, the repression was maintained at 0.3-fold that of vector control at even lower transfection quantities of 10 ng mP2 (Fig. 3B), a level of transfected DNA that would be unlikely to contribute non-specific effects on transcriptional readout. 3.3. mP2 represses the mP1-mediated basal regulation of the CYP3A4 and MDR1 gene The ability of the variant mP2 protein to repress the basal CYP3A4/Luc reporter readout stimulated by mP1 was assessed with the p3A4-13000 reporter construct. The transcriptional activity driven by mP1 was monitored against titrated quantities of co-transfected mP2 in Cos7 cells. At equimolar isoform transfection quantities, mP1 readout was reduced by 30%. At five molar mP2 excess, mP1 activity was reduced by 60% (Fig. 4). Further assessment revealed that at equimolar transfection quantities, mP2 repressed the mP1 readout on both the CYP3A4 reporters (p3A413000 and p3A4-362(7836/7208 ins) in HepG2 and Cos7 cell lines and on the p-7975(7012–1804) MDR1 reporter in Cos 7 cells (data not shown).

Pregnenolone-16␣-carbonitrile (PCN) is a potent mouse PXR ligand capable of marked induction of CYP3A genes (Robertson et al., 2003). The repressive potential of mP2 was evaluated by assessing the extent to which the mP1-mediated up-regulation of the CYP3A4-Luc reporter construct was affected by co-transfection of both isoforms into PCN treated cells. As expected, in HepG2 cells, PCN increased the mP1-mediated p3A4-362(7836/7298 ins) readout across a range of PCN concentrations (4–10 ␮M), however co-transfection of mP1 and mP2 at equimolar quantities indicated a similar reporter readout across the 4–10 ␮M PCN concentration range (data not shown). To investigate whether higher transfection quantities of mP2 could re-establish the repressive effect on PCN-induced mP1-mediated CYP3A4 readout, increasing amounts of mP2 were co-transfected across constant transfected mP1 quantities in PCN treated cells (Fig. 5). In the presence of 10 ␮M PCN, mP1-mediated p3A4-362(7836/7298 ins) transcription was repressed by 49 and 60% at 3:1 and 5:1 mP2:mP1 ratio, respectively. Similarly, in the presence of vehicle (DMSO) mP1 readout was reduced to 21% at 3:1 and 51% at 5:1 mP2:mP1 ratio (Fig. 5). 3.5. The repressive function of mP2 is not affected by inhibition of histone deacetylases To investigate whether the mP2 repressive function is mediated through the acetylation state of histones, transcriptional properties of mP2 were monitored in the presence of a histone deacetylase inhibitor trichostatin A (TSA). Following TSA treatment the repressive action of mP2 on the p3A4-13000 reporter readout was not relieved in Cos7 or HepG2 cells (data not shown). 3.6. Repressive action of mP2 operates independently of the PXR DNA-binding domain We next sought to investigate whether the mP2-mediated repressive action occurs through the ability of PXR protein to interact with CYP3A4 regulatory DNA-binding elements. Electrophoretic mobility shift assays (EMSAs) were used to analyze the DNA-binding of mP1 and mP2 to known PXR response elements (PXREs) within the CYP3A4 promoter and XREM regions. Initially we confirmed the expression vectors encoding the mPXR isoforms produced equivalent quantities of PXR protein following in vitro transcription/translation (Supp. Fig. 1A). To establish whether the two mPXR isoforms could bind CYP3A4 DNA-binding elements, two PXR associated DNA-binding elements retained in

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Fig. 5. mP2 represses the ligand-mediated P1 transcriptional response on the CYP3A4 gene. Transcriptional regulation of transiently co-transfected mP1, mP2 and V (indicated in ␮g) was evaluated on the p3A4-362 (7836/7208 ins) reporter construct in the presence of vehicle (DMSO) or PCN treatments (black and white bars, respectively) in HepG2 cells. Data are shown as fold vehicle treated vector control (V) readout and represent the mean of three independent experiments performed in triplicate. Error bars indicate SEM.

Fig. 6. mP2 repressive function is not mediated through DNA interactions. (A) Mouse PXR isoforms interact with PXREs within the CYP3A4 promoter. Interactions of mPXR isoforms with PXREs were evaluated using the electrophoretic mobility shift assay (EMSA). EMSAs were performed with in vitro translated RXR␣ (RXR) alone or in combination with mP1 or mP2 (indicated by “+”) and 32 P-labelled dNR1 or prPXRE oligonucleotide fragments (probes). DNA–protein complexes were resolved through 6% non-denaturing PAGE gels. Representative gels are shown. (B) Basal gene repression by mP2 is not mediated through DNA interactions. Transcriptional regulation of 0.1 ␮g of transiently transfected mXP2, mP1, mP2 and V expression constructs was evaluated on the CYP3A4-Luc [p3A4-13000 and p3A4-362 (7836/7208 ins)] reporters in HepG2 cells. Statistical analysis was performed in reference to V through application of the Student’s t-test. ***p ≤ 0.0001.

the p3A4-362 (7836/7208 ins) reporter were selected for comparative binding analysis (Supp. Fig. 1B). Both isoforms associated with each oligonucleotide in the EMSA as a heterodimer with RXR␣ (Fig. 6). Neither isoform was capable of binding the tested DNA elements in the absence of RXR␣ (data not shown). To test whether the repressive effect of mP2 is dependent on the mP2 ability to bind DNA within PXR response elements, the transcriptional capacity of a functionally viable chimeric mP2 (mXP2), which has its DNA-binding domain replaced by the GAL4-DBD (Kliewer et al., 1998) was investigated. Transcriptional analysis of mXP2 on the CYP3A4 reporter revealed mXP2 maintained a transcriptional activity comparable to, or below that of mP2 in HepG2 cells (Fig. 6B). This was also observed with the MDR1 reporter as well as in Cos7 cells (data not shown). Furthermore, analysis of the impact of mXP2 on mP1 action through utilization of cotransfection experiments revealed mXP2 maintained a repressive effect greater than or comparable to mP2 in HepG2 and Cos7 and in the presence of 10 ␮M PCN (data not shown). 3.7. PXR–protein partner interactions The potential for competitive protein interactions between mP1 and mP2 proteins were investigated next. Since RXR␣ is an obliga-

tory heterodimer partner of PXR for transactivation of target genes, the two isoforms were compared for differential RXR␣ association strength using GST pull-down assays (Fig. 7A). Initially, we confirmed the GSTmPXR fusion proteins and the [35 S]-methionine labelled candidate interaction proteins migrate at their predicted size when resolved with SDS-PAGE (data not shown). Subsequently, we observed GST-mP1 interacted most strongly with RXR␣, precipitating 41% of [35 S]-RXR␣ protein input while GST-mP2 showed significantly (p < 0.02) weaker interaction, precipitating only 18% of [35 S]-RXR␣ protein input (Fig. 7A). In addition, the potential of the two PXR isoforms to heterodimerize with each other was investigated (Fig. 7B). Heterodimerization between the mPXR isoforms was evident, showing consistent binding affinities with GST-mP1 precipitating 32% of mP2 input, and GST-mP2 precipitating 33% of mP1 input. 4. Discussion The discovery of PXR, its broad activation by therapeutic compounds and transcriptional regulation of a wide variety of genes involved in drug clearance, poses a novel explanation for inter-individual differences in response to treatment. Due to the predominant expression of hPXR434 in humans and mP1 (mPXR431 )

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Fig. 7. PXR isoforms display differential binding to partner proteins. GST pull-down assays with GSTmPXR fusion proteins were used to evaluate interactions of mPXR isoforms and RXR␣ proteins. (A) mPXR isoforms display differential binding to RXR␣ in unliganded state. GST pull-down assays were performed with GST-mP1 and GST-mP2 fusion proteins and [35 S] labelled in vitro translated RXR␣. Autoradiograph of SDS-PAGE gels following overnight exposure revealed protein bands of varying intensity, representing different degrees of interaction between GST-mP1 and GST-mP2 to RXR␣. Interaction of each RXR␣ band was quantified and expressed as a percentage of the original [35 S] input. The strongest interaction occurred between GST-mP1 and RXR␣, pulling down 41% of the total RXR␣ input, while 18% was pulled down with GST-mP2. GST-0 showed negligible binding 1% to RXR␣. (B) mP1 and mP2 heterodimerize. GST pull-down assays were performed with GST-mP1 and GST-mP2 fusion proteins and [35 S] labelled in vitro translated mP1 or mP2. Autoradiograph of SDS-PAGE gels revealed strong protein bands of similar intensity with GST-mP1 pulling down 32% of the [35 S]-mP2 (Bi) and GST-mP2 pulling down 33% [35 S]-mP1 (Bii). Representative gels are shown. Error bars indicate SEM. Statistical analysis was applied through the use of the Student’s t-test. *p ≤ 0.02.

in mice, studies have focused on elucidating their function, especially in mediating inductive drug interactions. Consequentially few studies have been directed towards understanding other PXR isoforms. Besides their initial characterization and subsequent quantification in human livers, little is known about the function of variant human PXR isoforms. Comparison of the activation profiles of mouse PXR isoforms revealed that mP2 (mPXR171–211 ) had a restricted range of ligands compared with mP1 (Kliewer et al., 1998). The current work examining the function of mP2 indicates that it may have a biological role distinct from mP1. Initially, the presence of mP2 and mP1 was detected in mouse liver, intestine and kidney (Fig. 2), consistent with previous studies (Kliewer et al., 1998). Human orthologues of mP2, the hPXR174–210 and hPXR174–214 isoforms, have also been identified in human liver tissue, supporting the notion that generation of mP2 is a conserved splicing event (Lamba et al., 2004). Furthermore, the relative abundance of mP2 and the variant human PXR mRNAs indicate that they are not rare transcripts. Thus, it would be interesting to determine the level of the corresponding proteins for PXR isoforms in

various tissues to assess the potential contribution they make to the biological action of PXR. During preparation of the current paper, the Shuetz group reported that hPXR174–210 has very similar properties to what we observe with the mouse PXR isoform (Lin et al., 2009). In addition to showing that PXR.2 (i.e. hPXR174–210 ) could reduce basal reporter readout, the presence of PXR.2 interfered with PXR.1-stimulated reporter induction. Furthermore, hPXR174–210 lacked activator binding capability in the presence or absence of the human PXR ligand rifampin and exhibited a strong ligand independent corepressor association. The repressive action of PXR.2 also occurred in the absence of the PXR DNA-binding domain (Lin et al., 2009). The Shuetz group went on to explore the in vivo relevance of their findings by assessing the impact on endogenous target genes in cell lines stably transfected with PXR.1 and PXR.2 expression constructs. The basal level of CYP3A4, P-gp and UGT1A1 mRNAs was reduced in the presence of PXR.2 (Lin et al., 2009). Therefore the repressive effect of PXR.2 can be exerted on bone fide endogenous genes as well as on co-transfected reporters. Furthermore, PXR

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−/− null mice have increased Cyp3A11 mRNA levels (Staudinger et al., 2001), suggesting PXR may have a repressive role in vivo by regulating the basal expression of target genes in the absence of ligand. Our data demonstrate that mP2 reduces the basal transcription of CYP3A4 and MDR1. Furthermore, mP2 represses the regulatory effects of mP1 on these target genes through a mechanism that may be independent of DNA-binding and histone deacetylation state. The current study, along with a recent study characterizing a human PXR isoform with an additional 39 amino acids at its N-terminal (Tompkins et al., 2008) and the recent work on hPXR174–210 (Conde et al., 2008; Lin et al., 2009) are the only functional reports on PXR isoforms. Such evidence of distinct PXR isoform actions indicates that they can play an important role in target gene modulation, both individually and collectively. Our observations were deduced from reporter-based assays using constructs representing two of the most significant PXR target genes, CYP3A4 and MDR1. On these reporters, we observed the basal transcriptional regulation by the two isoforms differed. As expected, mP1 raised the basal transcriptional readout of the MDR1 and CYP3A4 reporter constructs (Geick et al., 2001; Goodwin et al., 1999). In contrast, mP2 repressed the readout of the CYP3A4 and MDR1 reporter constructs. The repression was initially observed on the full-length CYP3A4 enhancer/promoter and was retained on the minimal CYP3A4 and MDR1 enhancer/proximal promoter regulatory regions, over a range of mP2 transfection quantities (Fig. 3) indicating either (i) the repressive mechanism occurs via interactions between the mP2 protein and DNA-binding elements within these regulatory regions or (ii) the repression occurs through specific protein–protein interactions, independent of gene regulatory regions. In addition to acting alone to reduce CYP3A4 and MDR1 transcription, mP2 has repressive action on basal and ligand-induced regulation of the CYP3A4 gene by mP1 (Figs. 4 and 5, respectively). Equimolar mP2 quantity was sufficient to repress the mP1-mediated basal CYP3A4 readout to 80% that of mP1 alone. Increasing the mP2 transfection quantity intensified the repressive action of mP2 indicating this effect was mP2 dependent. While mP2 could exert repression on the full-length CYP3A4 regulatory region, it also maintained a repressive effect on the essential CYP3A4 gene regulatory regions comprising the XREM adjacent to the promoter [present within the p3A4-362 (7836/7208 ins) reporter] indicating excluded upstream sequences and the spatial segregation of the CYP3A4 gene regulatory regions were not necessary for the mP2-mediated repression. Interestingly, mP2 was incapable of repressing the ligandinduced mP1 readout at equimolar isoform quantities over a range of concentrations for the mouse PXR-specific ligand (PCN). However, increased transfection quantities of mP2 were capable of repressing the PCN-induced CYP3A4 readout in the presence of mP1 (Fig. 5), indicating mP2 maintains a degree of repression for ligand-activated mP1. This suggests a dynamic balance of the relative PXR isoform abundance together with presence of ligands that may impact on the net action of PXR isoforms in regulating target genes. Despite the fact that mP2 mRNA has been shown to be <30% of the total PXR transcript, post-translational modifications of the PXR protein (Ding and Staudinger, 2005), interactions of PXR with protein partners, and altered protein stability/degradation could increase the effectiveness of repressive function exhibited by mP2. In addition, such factors could individually or collectively render mP1 transcriptional ability inactive, allowing mP2 to functionally dominate. At the time PXR was discovered it was found that mP2 and mP1 shared specificity for the same DNA-binding sites (Kliewer et al., 1998). Therefore, it is possible that mP2 could be exerting its repressive effect through interacting with PXREs in such regions of

target genes. A comparison of the DNA-binding ability of the two isoforms revealed mP2 associates with the PXR DNA-binding sites located within the enhancer and the proximal promoter elements of the CYP3A4 gene (Fig. 6A). This suggests potential for competitive, or independent DNA-binding events on the PXREs essential for PXR-mediated transcriptional up-regulation of CYP3A4. However, replacement of the DBD of mP2 with that of GAL4, thereby eliminating the ability of mP2 to bind these DNA elements, did not relieve its repressive function (Fig. 6B). These observations suggest the repressive action of mP2 is mediated independently of the native mPXR DNA-binding domain. The dependence on DNAbinding for repressive action of the human PXR.2 isoform was similarly explored by replacing its DBD with that of GAL4-DBD (Lin et al., 2009). While PXR.1-GAL4-DBD enhanced expression, the PXR.2-GAL4-DBD fusion protein reduced reporter readout. To further explore the repressive mechanism of mP2 we turned to potential competitive effects in protein–protein interactions known to play a role in PXR function. In accordance with previous studies (Goodwin et al., 1999; Kliewer et al., 1998), we observed that PXR associates with its DNA-binding sites only as a heterodimer with retinoid X receptor (RXR) (Fig. 6A). Our in vitro analysis of PXR-RXR protein interactions showed mP2 could associate with RXR␣ (Fig. 7A), suggesting mP2 may interfere with mP1 binding to RXR␣. We also observed that mouse PXR isoforms interact, thus generating mP1–mP2 heterodimers (Fig. 7B). The relevance of these findings is highlighted by a recent study demonstrating that AF2 domain mobility, co-activator interaction and PXR activation of target genes are dependent on the formation of a PXR:RXR␣ tetramer through association of PXR:PXR homodimers (Noble et al., 2006; Teotico et al., 2008). Interactions between mP2 and mP1 may lead to neutralization of mP1 action by disruption of essential protein–protein interactions (Fig. 7B). Whether such associations between PXR isoforms are representative of in vivo systems, or whether the affinities of PXR isoforms for specific proteins alter with changes in cellular states or post-translational modifications to either PXR isoform, RXR␣ itself, or other potential interacting protein complexes remains unanswered. Nuclear receptor isoforms have been identified for a wide range of nuclear receptors (Benoit et al., 2006; Dahlman-Wright et al., 2006; Flamant et al., 2006; Germain et al., 2006a,b; Keightley, 1998; Lu et al., 2006; Michalik et al., 2006; Moore et al., 2006). Closely related members of the NR1I nuclear receptor sub-family comprising of VDR, CAR and PXR have a number of alternate transcripts. CAR isoforms generally have an impairment or loss of function, such as an inability to translocate to the nucleus or bind DNA, ligand, co-regulators and obligatory heterodimer partner RXR (Lamba et al., 2005). The VDR isoform B1 exhibits distinct intra-nuclear speckled localization and relatively weaker transactivation activity compared to the parent isoform (Esteban et al., 2005; Sunn et al., 2005). Isoforms of other nuclear receptor families have distinct properties and functions. The presence of the ER isoform ER␤ has been found to have a protective role in breast cancer, as a consequence its expression is virtually non-existent in the majority of breast tumors (Bardin et al., 2004). Differential progesterone receptor (PR) and hepatocyte nuclear factor 4␣ (HNF4␣) isoforms levels are known to be altered in different tissues (Arnett-Mansfield et al., 2001; Bethea and Widmann, 1998; McGowan and Clarke, 1999; Niehof and Borlak, 2008; Oshima et al., 2007). In addition, a number of nuclear receptors including VDR, PR, HNF4␣ and the glucocorticoid receptor (GR) posses isoforms which can modulate the action of their alternate counterparts (Duma et al., 2006; Ebihara et al., 1996; Oakley et al., 1999; Pascussi et al., 2007; Tung et al., 1993). It is clear that the existence of nuclear receptor isoforms adds additional levels of complexity of nuclear receptor function. In summary, the variant mouse PXR isoform mP2 has a distinct repressive function in the basal and inductive regulation of

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drug detoxification and transport genes by the predominant form of mPXR found in the liver. The ability of mPXR171–211 to interfere with the action of the major PXR isoform provides another level of regulation over xenobiotic clearance pathways. While the current work attributes a repressive action to mP2 which operates at the gene regulatory level, further studies are required to determine whether variant PXR isoforms affect drug clearance pathways by altering levels of enzymes and transport proteins or their corresponding activities. In addition, it would be interesting to understand the potential factors (e.g. hormones, disease states, drugs) responsible for generating different levels of the variant PXR isoforms as well as impacting on their mode of action. Acknowledgements We would like to thank Prof. Oliver Burk for providing the 7975(7012–1804) reporter construct. Prof. Carsten Carlberg (University of Luxemburg, Luxembourg) for the pSG5hRXR␣ expression construct. pSG5–GAL4–PXR.2LBD was a kind gift from Prof Steve Kliewer (University of Texas Southwestern Medical Centre, Dallas, U.S.A.). Thank you to Prof Chris Liddle (Westmead Millennium Institute, Australia) for providing the CYP3A4 p3A4-13000 and the p3A4-362 (7836/7208 ins) constructs. We extend our gratitude to Dr. Andre Mahns, and the ANZAC Research Institute staff for their technical assistance. This work was supported by Australian NHMRC Project grant #352419 to GRR. M. Matic is a recipient of an Australian Postgraduate Award. A. Corradin is a Cancer Institute NSW Research Scholar and a recipient of an Australian Postgraduate Award. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biocel.2010.01.001. References Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophotonics Int 2004;11:36–42. Arnett-Mansfield RL, deFazio A, Wain GV, Jaworski RC, Byth K, Mote PA, et al. Relative expression of progesterone receptors A and B in endometrioid cancers of the endometrium. Cancer Res 2001;61:4576–82. Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of ERbeta expression as a common step in estrogen-dependent tumor progression. Endocr Relat Cancer 2004;11:537–51. Benoit G, Cooney A, Giguere V, Ingraham H, Lazar M, Muscat G, et al. International union of pharmacology. LXVI. Orphan nuclear receptors. Pharmacol Rev 2006;58:798–836. Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, et al. Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci USA 1998;95:12208–13. Bethea CL, Widmann AA. Differential expression of progestin receptor isoforms in the hypothalamus, pituitary, and endometrium of rhesus macaques. Endocrinology 1998;139:677–87. Blumberg B, Sabbagh Jr W, Juguilon H, Bolado Jr J, van Meter CM, Ong ES, et al. SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 1998;12:3195–205. Conde I, Lobo MV, Zamora J, Perez J, Gonzalez FJ, Alba E, et al. Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha. BMC Cancer 2008;8:174. Dahlman-Wright K, Cavailles V, Fuqua SA, Jordan VC, Katzenellenbogen JA, Korach KS, et al. International union of pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 2006;58:773–81. Ding X, Staudinger JL. Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C. Biochem Pharmacol 2005;69:867– 73. Dotzlaw H, Leygue E, Watson P, Murphy LC. The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue. Clin Cancer Res 1999;5:2103–7. Duma D, Jewell CM, Cidlowski JA. Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification. J Steroid Biochem Mol Biol 2006;102:11–21. Ebihara K, Masuhiro Y, Kitamoto T, Suzawa M, Uematsu Y, Yoshizawa T, et al. Intron retention generates a novel isoform of the murine vitamin D receptor that acts

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