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Role of Human Pregnane X Receptor in Tamoxifen- and 4-Hydroxytamoxifen-Mediated CYP3A4 Induction in Primary Human Hepatocytes and LS174T Cells

Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati Medical Center, Cincinnati, Ohio

(Received September 3, 2007; accepted February 15, 2008)

	Abstract

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Previously we observed that the antiestrogens tamoxifen and 4-hydroxytamoxifen (4OHT) induce CYP3A4 in primary human hepatocytes and activate human pregnane X receptor (PXR) in cell-based reporter assays. Given the complex cross-talk between nuclear receptors, tissue-specific expression of CYP3A4, and the potential for tamoxifen and 4OHT to interact with a myriad of receptors, this study was undertaken to gain mechanistic insights into the inductive effects of tamoxifen and 4OHT. First, we observed that transfection of the primary cultures of human hepatocytes with PXR-specific small interfering RNA reduced the PXR mRNA expression and the extent of CYP3A4 induction by tamoxifen and 4OHT by 50%. Second, in LS174T colon carcinoma cells, which were observed to have significantly lower PXR expression relative to human hepatocytes, neither tamoxifen nor 4OHT induced CYP3A4. Third, N-desmethyltamoxifen, which did not induce CYP3A4 in human hepatocytes, also did not activate PXR in LS174T cells. We then used cell-based reporter assay to evaluate the effects of other receptors such as glucocorticoid receptor GR and estrogen receptor ER on the transcriptional activation of PXR. The cotransfection of GR in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ER inhibited PXR-mediated basal activation of CYP3A4 promoter, possibly via competing for common cofactors such as steroid receptor coactivator 1 and glucocorticoid receptor interacting protein 1. Collectively, our findings suggest that the CYP3A4 induction by tamoxifen and 4OHT is primarily mediated by PXR but the overall stoichiometry of other nuclear receptors and transcription cofactors also contributes to the extent of the inductive effect.

With regard to drug-drug interactions of tamoxifen, the most prominent occurrences were observed in clinical trials in which tamoxifen coadministration was associated with increased clearance of letrozole and anastrozole (Dowsett et al., 1999, 2001). To better understand the causes of intersubject variability in tamoxifen metabolism and its apparent influence on the clearance of these aromatase inhibitors, in an earlier study we investigated the effect of tamoxifen on the expression and activity of CYP3A4. Using primary human hepatocytes, we observed that tamoxifen and 4OHT markedly increase the activity and expression of CYP3A4. Furthermore, we observed that both compounds activated the human pregnane X receptor (PXR) in cell-based reporter assays (Desai et al., 2002). It is well established that in addition to PXR, several other tissue-specific factors and nuclear receptors impact the transcriptional regulation of CYP3A4 (Pascussi et al., 2003a,b; Tegude et al., 2007). As such, the expression of PXR and other receptors, such as glucocorticoid receptor (GR) , and transcriptional factors, such as hepatic nuclear factor (HNF) 4, impact the regulation of PXR target genes (Pascussi et al., 2000; Li and Chiang, 2006).

This study was undertaken to gain mechanistic insights into the role of PXR and the impact of other tissue-specific transcription factors in the induction of CYP3A4 by tamoxifen and 4OHT. This investigation is particularly pertinent for these compounds because they are known to interact with various steroidal nuclear receptors, especially estrogen receptor (ER) . Furthermore, in an earlier study using Sprague-Dawley rats, p.o. administration of tamoxifen did not induce intestinal CYP3A4, suggesting that tissue-specific factors impact the CYP3A4 induction by tamoxifen (Cotreau et al., 2001). Our overall experimental strategy included 1) assessment of tamoxifen-mediated CYP3A4 induction in primary culture of human hepatocytes after down-regulating PXR in these cells by PXR-specific small interfering RNA (siRNA), 2) determination of CYP3A4 induction in LS174T colon carcinoma cells, and 3) evaluation of the effects of GR and ER on modulation of transcriptional activity of PXR by tamoxifen and 4OHT.

	Materials and Methods

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Chemicals and Reagents. Tamoxifen, 4OHT, rifampicin, and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO). NDMT was a gift from Dr. Frederick Beland at the Food and Drug Administration (Bethesda, MD).

Plasmids. The PXR expression plasmid pSG5-PXR-ATG, the CYP3A4 promoter plasmid [CYP3A4 proximal promoter bases -362 to +53 linked to the distal xenobiotic response element module (XREM) region] pGL3-CYP3A4 XREM-tk-luc, and steroid receptor coactivator 1 (SRC1) expression plasmid pSG5-FL-SRC1 were obtained from Dr. Bryan Goodwin (Goodwin et al., 1999). GR expression plasmid pSG5-hGR and GR response element reporter pGL3-(GRE)2-luc were provided by Dr. John Cidlowski (Oakley et al., 1999); glucocorticoid receptor interacting protein 1 (GRIP1) expression plasmid pSG5-HA-GRIP 1 was provided by Dr. Michael Stallcup (Ding et al., 1998); and ER expression plasmid pCMV-ER and ER response element (ERE) reporter plasmid pGL3-(ERE)3-luc were provided by Dr. Sohaib Khan (Singleton et al., 2003).

Cell Culture. Primary cultures of human hepatocytes as monolayers on collagen-coated plates (2 x 10⁶ cells/well of six-well plate) or cell suspensions were provided by the Liver Tissue Procurement and Distribution System (Pittsburgh, PA), which was funded by National Institutes of Health contract N01-DK-9-2310. LS174T and HepG2 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained as recommended in minimum essential medium and supplements, which were obtained from Invitrogen (Carlsbad, CA). The cells used in our study were within 7 passages after they were obtained from American Type Culture Collection at passage 100.

Transient Transfection Assays. Transient transfection of CYP3A4 promoter reporter plasmid and nuclear receptor expression plasmids in HepG2 and LS174T cells was performed using Lipofectamine and Plus reagents (Invitrogen) as described previously (Goodwin et al., 1999). Briefly, cells were plated in 24-well plates in minimum essential medium supplemented with delipidated fetal calf serum at a density of 1.2 x 10⁵ cells/well. Following 24 h of plating the cells, overnight transfections were performed using Lipofectamine and Plus reagents (Invitrogen) exactly as suggested by the manufacturer in the protocol for mammalian cells. Transfection mixes contained 75 ng of nuclear receptor expression vector, 75 ng of coactivators, 300 ng of luciferase reporter gene construct harboring promoter sequences of interest downstream of a luciferase reporter, and 300 ng of pCH110 (an expression vector containing β-galactosidase cDNA under T7 promoter) (Amersham, Piscataway, NJ). Equal quantities of DNA per transfection were maintained using an appropriate amount of empty pSG5 vector plasmid. Transfections with empty pSG5 vector were performed as negative controls. Following transfections, plasmid-containing medium was replaced with drug-containing medium and incubated for 24 to 48 h. The cell layers were washed twice with ice-cold phosphate buffer saline, pH 7.4, and scraped and collected in 250 µl of reporter lysis buffer provided with the β-galactosidase kit (Promega, Madison, WI). Cell lysates were used for determining protein content, luciferase enzyme activity using Luciferase assay system (Promega), and β-galactosidase activity by β-galactosidase assay kit (Promega). The luciferase activity was normalized to the β-galactosidase activity and expressed as -fold activation with respect to the solvent (0.1% dimethyl sulfoxide)-treated controls.

Transfection of siRNA in Primary Human Hepatocytes. Three sets of transfections (PXR siRNA, nontargeting siRNA, and mock transfections with Lipofectamine 2000) were performed in parallel in six-well plates. Lipofectamine 2000 and Williams' E medium supplemented with insulin-transferrin-selenium (Invitrogen) were used for transfecting 10 and 50 nM quantities of siRNA SmartPool duplexes (Dharmacon, Lafayette, CO) as per manufacturer's protocol for Lipofectamine 2000. The efficiency of this duplex pool was established in preliminary experiments using LS174T cells, wherein at concentrations ranging from 1 to 50 nM, siRNA duplexes were transfected in LS174T cells for 24 h. The knockdown of PXR was monitored in these transfected cells over 96 h and was found to gradually reduce to approximately 80% of controls at all the levels of duplex pool used. Based on these results, the transfections in human hepatocytes were allowed to proceed for 24 h, after which fresh medium was added to the transfected cells. Sixty hours post-transfections, drug treatments were initiated as described under Drug Treatment. At the end of 72 h of drug exposure, cells were dissolved in TRIzol and processed for RNA extraction. CYP3A4 and PXR-specific mRNA levels were then determined in drug-treated versus solvent-treated cells. Induction of CYP3A4 mRNA by test drugs in mock-transfected cells was considered maximal induction, and induction observed in cells treated with PXR-targeting and -nontargeting siRNA sequences was determined relative to the induction observed in mock-transfected hepatocytes. Cy-3 fluorescent-labeled RISC-free nontargeting siRNA siGLO (Dharmacon Inc.) was used in optimization of the transfection process.

Real-Time Polymerase Chain Reaction. Total RNA was treated with DNase I (DNA-free kit, Ambion, Austin, TX), and 2 µg was reverse-transcribed. The resulting cDNA was amplified and analyzed by real-time polymerase chain reaction (PCR) using ABI 7000 instrument (Applied Biosystems, Inc., Foster City, CA) with SYBR green detection. Following activation of Amplitaq Gold DNA polymerase, cDNA was amplified using cycling conditions: 10 min of initial denaturation at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C for primer annealing and extension. Primers: PXR (NM_003889 [GenBank] ) forward: 5'-GCA TCATCA GCT TTG CCA AAG-3', reverse: 5'-CCG CGT TGA ACA CTG TGT TG-3'; HNF4 (NM_178849 [GenBank] ) forward: 5'-AGC CTG CCC TCC ATC AAT G-3', reverse: 5'-CTC ACA CAC ATC TGC GAT GCT-3'; retinoid X receptor (RXR) (NM_002957 [GenBank] ) (Haugen et al., 2004) forward: 5'-GAG GCC TAC TGC AAG CAC AAG-3', reverse: 5'-CAG GCG GAG CAA GAG CTT AG-3';GR (NM_000176 [GenBank] ) (Raddatz et al., 2004) forward: 5'-CAA AAC TCT TGG ATT CTA TGC ATG AA-3', reverse: 5'-TTG GAA GCA ATA GTT AAG GAG ATT TTC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM_002046 [GenBank] ) forward: 5'-GAA GGT GAA GGT CGG AGT C-3', reverse: 5'-GAA GAT GGT GAT GGG ATT TC-3'; CYP3A4 (D11131 [GenBank] ) forward: 5'-CTT CAT CCA ATG GAC TGC ATA AAT-3', reverse 5'-TCC CAA GTA TAA CAC TCT ACA CAG ACA A-3' (Bowen et al., 2000); and ER (NM_000125 [GenBank] ) forward: 5'-CCA CCA ACC AGT GCA CCA TT-3', reverse: 5'-GGT CTT TTC GTA TCC CAC CTT TC-3' (de Cremoux et al., 2004).

The cDNA from HepG2 cells was serially diluted and amplified to generate relative standard curves for each mRNA under investigation. The amount of RNA in each reaction was calculated as per the starting concentration in the reverse-transcription reaction (2 µg). To standardize the amount of cDNA added to each reaction, the amount of the mRNA of interest was normalized to similarly calculated levels of GAPDH.

Drug Treatment. Cells in culture were treated with tamoxifen (1–10 µM), 4OHT (1–10 µM), NDMT (1–10 µM), rifampicin (10 µM), or phenobarbital (2 mM) for 72 h. Drug-containing medium was replaced every 24 h for the 72-h drug treatment period, after which cells were incubated with drug-free medium for 30 min to facilitate removal of drug from the cells. Cells were processed for the measurement of CYP3A4 activity, immunoreactive protein, and mRNA levels as described below. Cell viability was assessed in cell cultures maintained in 24-well plates following the drug treatment period using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay as described previously (Carmicheal et al., 1987).

The choice of tamoxifen and 4OHT concentrations used here was based on the clinical pharmacokinetics of tamoxifen. With a typical 20-mg b.i.d. regimen, tamoxifen plasma levels range from 0.1 to 1 µM, whereas the maximal 4OHT levels are 0.1 µM. However, the hepatic levels of these compounds may be 60-fold higher than that in serum (Lien et al., 1991). Plasma NDMT levels usually exceed tamoxifen levels at steady state (Kisanga et al. 2004).

Determination of CYP3A4 Activity. The rate of 6β-hydroxytestosterone formation by untreated control and drug-treated cells was used as a marker for CYP3A4 activity. Following drug treatment, cells in culture were incubated with media containing testosterone (250 µM) for 30 min. The media were collected and analyzed for testosterone, formed metabolites, and internal standard 11-hydroxyprogesterone (10 µg/ml) by reverse-phase high-performance liquid chromatography using 60% methanol as mobile phase (Desai et al., 2002).

Immunodetection of CYP3A4 and GR Protein. For CYP3A4 detection, cells were homogenized in HEPES/EDTA buffer and microsomes were prepared by differential centrifugation. For GR detection, the cells were lysed in radioimmunoprecipitation assay buffer (Pierce, Rockford, IL). The proteins were quantitated by Lowry assay using bovine serum albumin (BSA) standards. The proteins were treated uniformly and were maintained at 4°C until resolved to minimize degradation. Equal amounts of proteins (3 µg for CYP3A4, 20 µg for GR) were resolved using SDS-polyacrylamide gel electrophoresis (12% acrylamide) and transferred to nitrocellulose membranes. The membranes were then blocked with 3% BSA in Tween (0.1%)/phosphate-buffered saline, pH 7.4, for 45 min and then treated with primary anti-CYP3A4 antibody (BD Biosciences, San Jose, CA) or anti-GR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by horseradish peroxidase-conjugated anti-mouse or anti-goat secondary antibody (Sigma Chemical Co. and Santa Cruz Biotechnology). The protein bands were visualized using enhanced chemiluminescence detection (Amersham).

Northern Blot Analysis of CYP3A4 mRNA. Total cellular RNA was isolated using TRIzol (Invitrogen), quantitated spectrophotometrically, and 10 µg fractionated using electrophoresis, followed by overnight transfer onto a nylon membrane (Millipore, Bedford, MA). Isolated CYP3A4 cDNA probes (Oxford Biomedical Research, Oxford, MI) were labeled with ³²P-labeled dCTP (NEN, Boston, MA) using the random primer method and hybridized. The membranes were exposed to X-ray film, and the developed bands were quantitated using NucleoVision image analyzer (Nucleotech, San Mateo, CA). The probe used here, as well as testosterone 6β-hydroxylase activity, may not be specific for CYP3A4 and may also detect CYP3A5, which is typically a minor component of the overall CYP3A pool (Westlind-Johnsson et al., 2003).

Statistical and Data Analysis. The differences in CYP3A4 activity, immunoreactive protein content, mRNA levels, and cell viability in control versus treated groups were analyzed using a one-factor analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons or a t test for pair-wise comparisons at = 0.05.

	Results

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Role of PXR in Tamoxifen- and 4OHT-Mediated CYP3A4 Induction. We first evaluated the induction of CYP3A4 in primary cultures of human hepatocytes transfected with PXR-specific siRNA. Our initial attempts entailed optimization of the conditions for PXR siRNA transfection in LS174T cells using 10 to 50 nM PXR-specific siRNA duplexes that were able to down-regulate PXR mRNA up to 80% (data not shown). These attempts indicated that at a 10 nM concentration the siRNA sequences were noncytotoxic, but cytotoxicity was observed at a 50 nM concentration. As shown in Fig. 1A, PXR expression was reduced by 50% in cells transfected with 10 nM PXR siRNA, whereas no changes in PXR levels were apparent in mock-transfected and nontargeting siRNA-transfected hepatocytes. The extent of CYP3A4 induction (difference in the mRNA levels in drug-treated versus solvent-treated controls) was determined separately for mock siRNA-treated and PXR siRNA-treated hepatocytes. As seen in Fig. 1B, cells transfected with PXR siRNA, which exhibited a 50% decrease in the PXR mRNA levels, exhibited significantly lower extent of CYP3A4 induction than mock-transfected cells. The magnitude of CYP3A4 induction by tamoxifen, 4OHT, and rifampicin was reduced by 53, 77, and 52%, respectively. It is important to note that the extent of CYP3A4 induction in nontargeting siRNA-transfected cells was not significantly different from that in mock-transfected cells. In parallel, Cy-3-labeled siRNA (siGLO, Dharmacon) was transfected in primary human hepatocytes on a glass slide and viewed by confocal microscopy to ensure intracellular compartmentalization of the siRNA sequence, comparable in length with the silencing siRNA. In hepatocytes from the donors in which we did not observe any reduction of PXR mRNA, possibly because of lack of effective transfection in these cells, a change in CYP3A4 inducibility was not observed (data not shown). This observation corroborates that the change in extent of CYP3A4 inducibility was related to change in PXR expression.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Effect of PXR siRNA on PXR mRNA expression and CYP3A4 induction. A, PXR mRNA expression in primary human hepatocytes transfected with PXR-specific pool of 4 siRNA duplexes (10 nM), pool of nontargeting siRNA duplexes (10 nM), and transfecting reagent alone (mock-transfected) normalized to GAPDH mRNA expression. Data shown are mean ± S.D. from three donors of human hepatocytes, ANOVA, p < 0.05. B, extent of CYP3A4 induction after the primary human hepatocytes transfected with PXR siRNA were treated for 72 h with rifampicin (Rif, 10 µM), tamoxifen (Tam, 5 µM), and 4OHT (5 µM) and expressed as percentage of CYP3A4 induction achieved in mock-transfected hepatocytes.

We then determined the extent of PXR-mediated activation of CYP3A4 promoter by tamoxifen and 4OHT in HepG2 and LS174T cells. These cells were transiently transfected with a luciferase reporter construct harboring PXR-responsive enhancer module (XREM) and a PXR expression plasmid (pSG5-PXR-ATG) (Goodwin et al., 1999). Tamoxifen and 4OHT activated PXR in both LS174T and HepG2 cells (Fig. 2). In HepG2 cells, at 5 µM concentration, tamoxifen moderately activated PXR (5-fold), whereas 4OHT strongly activated PXR (10-fold), the magnitude of the latter being comparable with that of the prototypical PXR activator, rifampicin (10 µM). The overall magnitude of activation in LS174T cells was lower compared with that observed in HepG2 cells. For instance, at 5 µM concentration, the extent of activation in tamoxifen- and 4OHT-treated LS174T cells was 2.5- and 5-fold, respectively. NDMT did not activate PXR in HepG2 and LS174T cells.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Activation of PXR-mediated transcription at CYP3A4 promoter in HepG2 and LS174T cells. Cells were transiently transfected with the pSG5-PXR-ATG expression plasmid, pGL3-XREM-CYP3A4-luciferase reporter plasmid, and pCH110. Twenty-four hours post-transfection, cells were treated with phenobarbital, rifampicin (Rif), tamoxifen (Tam), 4OHT, and NDMT for 24 h. -Fold increase in the luciferase reporter activity normalized to β-galactosidase activity following drug treatment is depicted [n = 3 separate experiments, mean ± S.D.; *, statistically significant increase (p < 0.05) in normalized luciferase activity on drug treatment as compared with respective untreated HepG2 or LS174T controls].

Effect of NDMT on Hepatic CYP3A4. In our previous study, we investigated the effects of tamoxifen and 4-OHT using four batches of human hepatocytes (Desai et al., 2002). In this report we have included data from additional donors of human hepatocytes in which the effect of these agents and NDMT on the expression and activity of hepatic CYP3A4 was evaluated. First, differences in the rate of testosterone 6β-hydroxylase activity of cells treated with vehicle (0.1% dimethyl sulfoxide) alone compared with those treated with the antiestrogens or prototypical inducers rifampicin and phenobarbital were evaluated as a marker of CYP3A4 activity. Protein and mRNA levels were evaluated by Western blotting and real-time PCR, respectively. As indicated in Table 1, at concentrations up to 10 µM, NDMT-treated hepatocytes did not exhibit an increase in CYP3A4 activity, protein, or mRNA levels compared with solvent-treated cells. NDMT, although structurally very similar to tamoxifen, did not activate PXR in HepG2 and LS174T (Fig. 2), which is consistent with the lack of its effect on CYP3A4 expression.

Expression of Nuclear Receptors in Hepatocytes and LS174T Cells. The expression of PXR, HNF4, RXR, ER, and GR were compared in HepG2 and LS174T cell lines with primary human hepatocytes. As shown in Fig. 3, although the overall levels of these transcription factors were lower in HepG2 and LS174T cells, the levels of PXR and the other key CYP3A4 regulator, GR, were strikingly lower in the cell lines. Most notably, GR was undetectable in LS174T cells, whereas its expression levels were easily detectable in HepG2 cells and primary hepatocytes. This observation was further confirmed by assessing the GR protein levels in these cells using Western blotting (Fig. 4). Because tamoxifen and 4OHT are ER ligands, we assessed the levels of ER in these cells. In primary hepatocytes, ER was expressed at levels significantly lower than those in ER-positive MCF-7 breast cancer cells. Its expression in HepG2 and LS174T cells was 5- and 10-fold lower, respectively, compared with hepatocytes. Given this reduced expression of GR and ER in LS174T cells, we investigated the role of these nuclear receptors in human PXR-mediated transactivation of CYP3A4 reporter by the antiestrogens.

View larger version (14K): [in this window] [in a new window]   FIG. 3. mRNA expression of nuclear receptors. Reverse transcription followed by real-time PCR was performed on RNA isolated from primary human hepatocytes (mean from three separate donors) and HepG2, LS174T, and MCF-7 cells. mRNA levels of various transcription factors normalized to GAPDH expression are shown. HNF4 and PXR (A), GR and RXR (B), and ER (C).

View larger version (7K): [in this window] [in a new window]   FIG. 4. Expression of GR protein. Western blot depicting immunoreactive protein levels of GR protein expressed in primary human hepatocytes, HepG2, and LS174T cells. Cell lysates (20 µg) were resolved using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then blocked with 3% BSA in Tween (0.1%)/phosphate-buffered saline, pH 7.4, and then treated with primary anti-GR antibody followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody. The protein bands were visualized using enhanced chemiluminescence detection.

GR Potentiated Tamoxifen/4OHT-Mediated PXR Activation.

View larger version (10K): [in this window] [in a new window]   FIG. 5. Effect of GR on transcription of CYP3A4 promoter. LS174T cells were transiently transfected with GR expression plasmid and/or PXR expression plasmid, pGL3-XREM-CYP3A4-luc reporter plasmid, and β-galactosidase expression plasmid. The transfected cells were treated for 24 h with rifampicin (Rif, 10 µM), tamoxifen (Tam, 5 µM), and 4OHT (5 µM), and luciferase activity of these cells was normalized to the β-galactosidase activity. -Fold increase of normalized luciferase activity over untreated control cells is depicted (n = 3 separate experiments, mean ± S.D., paired t test between PXR only and PXR + GR expressed cells, *, p < 0.05).

View larger version (11K): [in this window] [in a new window]   FIG. 6. Effect of ER on repression of PXR-mediated transcription of CYP3A4 promoter. LS174T cells were transiently cotransfected with pGL3-XREM-CYP3A4-luc reporter plasmid, β-galactosidase plasmid, PXR expression plasmid, with or without pCMV-ER expression plasmid. The transfected cells were then treated with estradiol (1 nM) for 24 h. β-Galactosidase-normalized luciferase activity in the presence and absence of ER/activated ER was compared (n = 3 separate experiments, mean ± S.D., paired t test, *, p < 0.05).

View larger version (11K): [in this window] [in a new window]   FIG. 7. Effect of coactivators on repression of PXR-mediated transcription of CYP3A4 promoter by ER. LS174T cells were transiently cotransfected with PXR expression plasmid, pGL3-XREM-CYP3A4-luc reporter plasmid, and pCH110 β-galactosidase plasmid. Additionally, in parallel, cells were also transfected with increasing concentrations of ER expression plasmid (5 and 37.5 ng). From cells transfected with the highest quantity of pCMV-ER expression plasmid (37.5 ng), some were also transfected with increasing amounts of coactivators SRC1 (pSG5-FL-SRC1) and GRIP1 (pSG5-HA-GRIP1) (both 5 and 37.5 ng). Transfections were treated with 1 nM estradiol for 24 h for ER activation. Luciferase activity was normalized to β-galactosidase activity (n = 3 separate experiments, mean ± S.D., ANOVA, Tukey's multiple comparison test, *, p < 0.05).

Effect of Tamoxifen and 4OHT on CYP3A4 Expression in LS174T Cells. We also evaluated the effects of tamoxifen and 4OHT on CYP3A4 in LS174T cells, a cell culture model previously used for assessing effects on intestinal drug-metabolizing enzymes (Schuetz et al., 2000). Notably, neither tamoxifen nor 4OHT at concentrations ranging from 1 to 10 µM increased testosterone 6β-hydroxylase activity in LS174T cells, although the prototypical inducers, rifampicin and phenobarbital, increased CYP3A4 activity in these cells by 1.97- and 5.3-fold, respectively (t test, p < 0.05) (Table 2).

The effect of the antiestrogens on the amount of CYP3A4-specific mRNA and CYP3A4-immunoreactive protein in LS174T cells is summarized in Table 2. Neither tamoxifen nor 4OHT had any significant influence on CYP3A4 mRNA levels in LS174T cells, whereas 1.89- and 4.12-fold increases were observed with rifampicin and phenobarbital treatment, respectively. This observation is in contrast to our observations in human hepatocytes, in which we observed a marked dose-dependent increase in mRNA levels in hepatocytes treated with tamoxifen and 4OHT. We also determined the CYP3A4 mRNA levels using the more sensitive technique of real-time PCR. Tamoxifen and 4OHT both exhibited a very low 2- and 3-fold increase in CYP3A4 mRNA in LS174T cells, respectively, which was in striking contrast compared with the robust 17-fold increase observed on treatment with rifampicin. Also in the case of protein levels, whereas rifampicin and phenobarbital increased the CYP3A4 protein levels significantly (paired t test, p < 0.05), tamoxifen and 4OHT did not show significant change.

	Discussion

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We have previously shown that tamoxifen and 4OHT induce CYP3A4 in primary human hepatocytes and activate PXR in HuH7 cells (Desai et al., 2002). In this study we have further probed the role of PXR and other receptors that may modulate the expression of CYP3A4 or are known targets for tamoxifen and 4OHT. First, we compared the extent of CYP3A4 induction in primary hepatocytes and PXR activation in HepG2 and LS174T cells by tamoxifen, 4OHT, and NDMT. In general, there was a good agreement between the CYP3A4 mRNA induction in primary human hepatocytes and PXR activation in transfection assays; the order of magnitude of PXR activation (i.e., 4OHT > tamoxifen > NDMT) matched the order of CYP3A4 induction in human hepatocytes. The critical role of PXR in antiestrogen-mediated CYP3A4 induction was further shown by using siRNA targeting PXR mRNA. Attenuation of PXR expression in primary human hepatocytes with siRNA caused a marked reduction in the overall extent of CYP3A4 induction by tamoxifen and 4OHT. For example, with a 50% reduction in PXR mRNA levels in hepatocytes, we observed approximately 50 to 70% reduction in the magnitude of CYP3A4 induction by tamoxifen, 4OHT, and rifampicin, a prototypical CYP3A4 inducer and PXR activator. Previously Pascussi et al. (2000) have shown that CYP3A4 inducibility is dependent on PXR expression as evident from decreased CYP3A4 inducibility on cytokine-mediated down-regulation of PXR. Other studies assessed CYP3A4 inducibility in hepatocytes transfected with dominant negative PXR receptor (Kocarek et al., 2002) and in PXR null and transgenic mice (Xie et al., 2000). Here, we used a novel approach that entailed attenuation of PXR by siRNA to underscore the importance of PXR expression on CYP3A4 induction by tamoxifen and 4OHT in primary human hepatocytes.

We next assessed the potential role of other transcription factors in the CYP3A4-inductive effects of tamoxifen and 4OHT. Previous studies have suggested a role of GR in CYP3A4 induction by xenobiotics. For example, the GR agonist dexamethasone induces CYP3A4, an effect blocked by the antiglucocorticoid mifepristone (Ogg et al., 1999; Matsunaga et al., 2004). Rifampicin, a potent CYP3A4 inducer and PXR activator, is able to transactivate the CYP3A4 promoter via GR in the absence of PXR cotransfection (El-Sankary et al., 2000), whereas cotransfection of GR and PXR expression plasmids is needed for maximal CYP3A4 promoter activation (Gibson et al., 2002). Furthermore, GR may have a role in the expression of PXR (Pascussi et al., 2000, 2001). Interestingly, the GR ligand dexamethasone increased CYP3A1/23 levels in rat livers, but it did not do so in rat intestines (Hartley et al., 2004). Although the role of GR in CYP3A4 induction awaits further clarification, it appears that its contribution is tissue- and ligand-specific. The presence of GR is critical and the inductive effects are GR-dependent for some ligands, whereas the inductive effects are GR-potentiated for other ligands (Ogg et al., 1999; Schuetz et al., 2000).

To evaluate the role of GR, in this study we first assessed the expression of GR and that of other nuclear receptors in primary human hepatocytes and HepG2 and LS174T cell lines. Although the levels of several transcription factors, including PXR, were lower in LS174T cells, the most striking difference was the absence of measurable GR expression in LS174T cells at both mRNA and protein levels. This may account for the observed lack of CYP3A4 induction by tamoxifen and 4OHT in LS174T cells. Further support for the role of GR is derived from transient transfection assays in which the effect of cotransfecting PXR and GR on CYP3A4 promoter was compared with transfection of PXR or GR alone. Although tamoxifen and 4OHT did not activate CYP3A4 transcription when cells were transfected with GR alone, cotransfection of GR with PXR significantly potentiated the transcription of CYP3A4 promoter transcription by tamoxifen and 4OHT. Collectively, our findings suggest that GR may be an important factor for maximal induction of CYP3A4 by tamoxifen and 4OHT. The plausible role of GR in regulation of PXR expression in LS174T cells that could result in CYP3A4 induction was not explored in this study (Pascussi et al., 2000).

Next, we examined the role of ER on the CYP3A4 promoter activation by tamoxifen and 4OHT. ER regulates the expression of many genes in a complex manner, resulting in either transcriptional activation or repression depending on the target gene. Given that tamoxifen and 4OHT are ER ligands, a role of these receptors mediating complex downstream pathways resulting in transactivation of CYP3A4 gene can be envisioned especially because CYP3A4 promoter harbors at least partial EREs (Hashimoto et al., 1993). As in the case with GR, on tamoxifen and 4OHT treatment, ER did not activate CYP3A4 promoter when transfected in absence of PXR. However, unlike GR, ER did not exhibit synergistic interaction with PXR at the CYP3A4 promoter in the copresence of PXR. In fact, when ER was cotransfected alone with CYP3A4-XREM-luc reporter plasmid, it appeared to repress PXR-mediated basal transcriptional activation of CYP3A4 promoter. The magnitude of the repression was considerably higher in the presence of physiologically relevant levels of estradiol. This is in contrast to findings of Mnif et al. (2007), who report that estradiol is capable of activating PXR. However, in that study concentrations of estradiol were 10- to 100-fold higher than those used in our study, and the reporter used did not harbor CYP3A4 promoter. We attributed the observed repressive effect of activated ER to competition for commonly required cofactors in transcriptional machinery such as SRC1 and GRIP1. Indeed, coexpression of these transcription cofactors was able to restore the basal level of transcription even in the repressive presence of activated ER.

Similar examples of competitive repression have been reported earlier. Repression of ER activity by CAR (Min et al., 2002) and thyroid receptor activity by ER has also been attributed to squelching of p160 coactivators SRC1 and GRIP1 (also called SRC2) or other downstream coactivators (Lopez et al., 2005). Our observations with respect to PXR and ER are analogous to these results. Furthermore, it is important to note that a direct involvement of ER in ligand-dependent transcriptional modulation is possible because the CYP3A4 promoter does harbor estrogen response element motifs (Hashimoto et al., 1993).

An interesting aspect of our findings is that tamoxifen and 4OHT may have the potential to induce CYP3A4 in a tissue-specific manner. An earlier study by Cotreau et al. (2002) suggested that tamoxifen administration may lead to induction of hepatic but not intestinal CYP3A in rats. Several other "nonclassical inducers" include dexamethasone, anti-HIV agents amprenavir and nelfinavir, efavirenz, and L-742694, which induce hepatic but not intestinal CYP3A (Huang et al., 2001; Mouly et al., 2002; Hartley et al., 2004). The lack of CYP3A4 induction by tamoxifen/4OHT in LS174T cells may be partly explained by the observed differences in the expression of nuclear receptors relative to hepatocytes. Because the presence of both hPXR and GR is required for maximal induction of CYP3A4 by tamoxifen/4OHT, lower levels of hPXR and absence of GR in LS174T cells may contribute to the lack of tamoxifen/4OHT-mediated CYP3A4 induction in these cells. Although caution is warranted in interpreting observations made using a transformed cell line, the lack of tamoxifen/4OHT-mediated CYP3A4 induction in LS174T cells may explain, at least partially, the lack of induction of intestinal CYP3A in rats. In humans, colonic GR is approximately 10-fold lower compared with that in liver (Pujols et al., 2002). A limitation of our study is that we did not explore the role of CAR and HNF4 in CYP3A4 transactivation in response to tamoxifen/4-OHT. In a previous study, Tirona et al. (2003) suggested a critical role of HNF4 in the PXR- and CAR-mediated transcriptional activation of CYP3A4 in Caco-2 cells. Furthermore, Tegude et al. (2007) underscored the role of HNF4 in directly regulating CYP3A4 in LS174T cells. Thus, it is likely that in addition to GR, reduced expression of other transcription factors, such as HNF4 observed in LS174T cells, and/or interaction with CAR may also contribute to the apparent lack of CYP3A4 induction by tamoxifen/4OHT in these cells.

In summary, our study shows that the induction of CYP3A4 by antiestrogens tamoxifen and 4OHT primarily entails transactivation of CYP3A4 by PXR, which is further potentiated by GR. It is noteworthy that although the prototypical CYP3A4 inducers rifampicin and phenobarbital induced CYP3A4 in the colon carcinoma cell line LS174T, tamoxifen and 4OHT did not do so. Thus, these agents may exhibit an atypical, tissue-dependent pattern of induction. Based on our study, the molecular bases for such tissue-specific CYP3A4 up-regulation may entail lower expression of PXR and GR in LS174T cells compared with human hepatocytes. It is evident that CYP3A4 induction depends on the overall stoichiometry of several key regulatory receptors, coactivators, and corepressors in a given tissue, which together govern the inductive effects of tamoxifen. Such findings have implications for induction of CYP3A4 and related genes by tamoxifen in nonhepatic tissues. For example, in breast carcinoma tissue, CYP3A4 induction may impact tamoxifen efficacy, whereas increased bioactivation in endometrial tissues may contribute to increased toxicity.

	Acknowledgments

 
We thank Stephen Strom, Ph.D., University of Pittsburgh, for valuable comments and suggestions during the course of this study.

	Footnotes

 
This study was supported in part by a grant from the American Cancer Society (Ohio Division) (P.B.D.) and a predoctoral fellowship (R.S.S.) from the Susan G. Komen for the Cure foundation.

No official support or endorsement of this article by the Food and Drug Administration is intended or should be inferred.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.018598.

ABBREVIATIONS: NDMT, N-desmethyltamoxifen; 4OHT, 4-hydroxytamoxifen; PXR, pregnane X receptor; GR, glucocorticoid receptor; HNF, hepatic nuclear factor; ER, estrogen receptor; siRNA, small interfering RNA; XREM, xenobiotic response element module; SRC1, steroid receptor coactivator 1; GRIP1, glucocorticoid receptor interacting protein 1; ERE, estrogen receptor response element; PCR, polymerase chain reaction; RXR, retinoid X receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BSA, bovine serum albumin; ANOVA, analysis of variance; CAR, constitutive androstane receptor.

¹ Current affiliation: Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT.

² Current affiliation: Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD.

Address correspondence to: Pankaj B. Desai, Division of Pharmaceutical Sciences, College of Pharmacy, University of Cincinnati Medical Center, 3225 Eden Avenue, Cincinnati, OH 45267-0004. E-mail: pankaj.desai{at}uc.edu

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