|Home | About | Journals | Submit | Contact Us | Français|
Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100bp sequence (−1000/−901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4α (HNF4α) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4α and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4α to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.
Pregnane X receptor (PXR), an orphan member of the nuclear steroid/thyroid receptor super-family, was first characterized as an activator of transcription of the CYP3A4 gene in response to various drugs and steroid metabolites (1). PXR has now been established as a key transcription factor that induces the hepatic capacity for xenobiotic metabolism and excretion by activating a group of genes that encode xenobiotic-metabolizing enzymes and transporters. In addition, PXR has recently been reported to regulate p38 mitogen-activated protein kinase-mediated cell migration signals by activating the GADD45β gene (2). The general mechanism underlying these activations is a direct binding of PXR to the response enhancers of its target genes. PXR has also been demonstrated to repress transcription of hepatic genes such as PCK1, G6Pase and CYP7A1, thereby regulating energy and cholesterol homeostasis (3–5). The repression of these genes appears to be regulated through interactions of PXR with other transcription factors and co-activators such as FOXO1, CREB and PGC1α, but not by direct DNA binding. However, the molecular mechanism by which PXR represses transcription remains elusive at the present time. Utilizing human estrogen sulfotransferase (SULT1E1) gene as a model, here we have investigated the molecular mechanism of PXR-mediated repression of gene expression.
Estrogen levels can be regulated by altering biosynthesis by aromatase, metabolic inactivation by sulfotransferase and re-activation by sulfatase. Although the actions of estrogen and the development of estrogen-related diseases by effects on aromatase are well established, estrogen metabolism is also involved in the regulation of estrogen activity. SULT1E1 exhibits the highest affinity for estrogens among known sulfotransferases and is the primary enzyme responsible for sulfation of β-estradiol and estrone. SULT1E1 is expressed at low levels in the livers of normal mice and is highly expressed in livers of diabetogenic db/db mice (6). Expression of SULT1E1 in adipose tissue of transgenic female mice revealed that SULT1E1 activity regulates fat and glucose homeostasis (7). Sult1e1−/− female mice were reported to develop placental thrombosis and spontaneous fetal loss (8). High estrogen sulfatase (STS) activity has been detected in tumor tissues of breast cancer patients and was correlated with a poor prognosis for breast cancer in women (9,10). Drugs and steroid hormones are known to induce SULT1E1 in mouse liver via activation of nuclear receptors such as constitutive active/androstane receptor (CAR), liver X receptor and glucocorticoid receptor (11–13).
In this present study, given that the SULT1E1 gene was repressed by rifampicin (RIF) in human primary hepatocytes, we utilized human hepatocellular carcinoma Huh7 and HepG2 cells to investigate the molecular mechanism of the PXR-mediated repression of the SULT1E1 gene.
First the repressive activity was delineated to a 100bp distal enhancer within the SULT1E1 promoter. Subsequently, chromatin immunoprecipitation (ChIP) and chromatin conformation capture (3C) assays were employed to identify hepatocyte nuclear factor 4α (HNF4α) and its binding site within the 100bp enhancer as the target of PXR to repress the SULT1E1 gene. Here, we present these experimental considerations to suggest a novel mechanism, in which a drug-activated PXR cross-talks with HNF4α, disrupting an active chromatin structure, and thereby repressing transcription of the SULT1E1 gene.
RIF, SR12813, cycloheximide (CHX), TCPOBOP, anti-FLAG-M2 HRP-conjugated antibody and FLAG-M2 agarose were purchased from Sigma-Aldrich (St Louis, MO, USA); CITCO from BioMol (Plymouth Meeting, PA, USA); restriction endonucleases and DNA-modifying enzymes from New England Biolabs, Inc. (Beverly, MA, USA); antibody to human PXR from Perseus Proteomics Inc. (Tokyo, Japan); mouse, goat and rabbit normal IgGs, antibodies against HNF3β (M-20), HNF4α (H-171) and β-actin (C4) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-V5 HRP-conjugated antibody from Invitrogen (Carlsbad, CA, USA); anti-H3K9K14ac (06–599) from Millipore (Billerica, MA, USA); [32P]dATP from GE Healthcare (Piscataway, NJ, USA) and [35S]methionine from PerkinElmer (Waltham, MA, USA). ON-TARGETplus SMART pool HNF4α (catalog number L-003406-00-0005), HNF3β (catalog number L-010089-00-0005), PGC1α (catalog number L-005111-00-0005) and ON-TARGETplus siCONTROL non-targeting pool (catalog number D-001810-10) were purchased from Dharmacon Research (Lafayette, CO, USA). All primers, oligonucleotides and real-time PCR probes used for present experiments are listed in Supplementary Table S1–S4.
In all vectors, the prefixes h and m in front of inserts denote human and mouse, respectively. pCR3/hPXR, pCR3/FLAGhPXR, pcDNA3.1/hHNF4α, XREM-3A4-Luc, pCR3/mCART176V, pGL3/1.8 kbp-2B6-Luc, adeno-hPXR and adeno-β-gal have been described previously (4,14–16). cDNA encoding the full-length human HNF3β was amplified with appropriate PCR primers and cloned into pcDNA3.1/V5-His-TOPO (Invitrogen) to yield pcDNA3.1/hHNF3β.
The 5.3 and 1.1kb DNA fragments containing the 5′-flanking region of human SULT1E1 gene were amplified from human genomic DNA (Promega, Madison, WI, USA) using appropriate pairs of PCR primers. Amplified DNA fragments were cloned into KpnI/XhoI-digested pGL3-Basic vector (Promega) to yield pGL3/5.3 kb-hSULT1E1 and pGL3/1.1 kb-hSULT1E1, respectively. The 100-bp enhancer region was amplified from pGL3/5.3 kb-hSULT1E1 using appropriate pairs of PCR primers and cloned into KpnI/XhoI-digested pGL3-Basic vector containing a 160bp thymidine kinase promoter to yield pGL3/hSULT1E1enhancer-TK (17). Each of the mutant promoter constructs was produced by site-directed mutagenesis using appropriate oligonucleotides. To construct Luc reporter constructs for 3C assays, a double-stranded oligonucleotide 5′-GATCACTAGCAAAATAGGCTGTCCCCAGA-3′ encoding RV primer 3 was inserted into the BglII-digested site of each Luc reporter constructs, and then the original sequence of RV primer 3 in the pGL3-Basic was deleted by site-directed mutagenesis.
Human hepatocellular carcinoma Huh7 cells were maintained in minimal essential medium (MEM) supplemented with 10% FBS, 2mM l-glutamine, penicillin (100 U/ml) and streptomycin (100µg/ml) in an atmosphere of 5% CO2 at 37°C. For Luc reporter assays, Huh7 cells were transiently transfected with Firefly luciferase reporter construct along with pCR3/hPXR or pCR3/mCART176V, using FuGene 6 (Roche Applied sciences, Indianapolis, IN, USA). pRL-TK (Promega) was co-transfected as control. The final amounts of transfected DNAs were adjusted to constant levels by adding pCR3 (Invitrogen) empty vector. Twenty-four hours after transfection, the cells were treated with a given drug in FBS-free MEM for an additional 24h and luciferase reporter assays were performed as previously described (3). For adenoviral infection, cells were cultured in MEM medium containing adeno-β-gal or adeno-hPXR at a multiplicity of infection of 10. After incubation for 30h, these cells were treated with either RIF (10µM) or SR12813 (3µM) in FBS-free MEM for a given time. For siRNA knockdown, trypsinized cells were reverse transfected with siRNA (50pmol) in MEM medium, using Lipofectamine 2000 (Invitrogen). After incubation for 24h, culture medium was replaced with FBS-free MEM and cells were incubated for another 16h. For real-time PCR, trypsinized Huh7 cells were reverse transfected with pCR3 or pCR3/mCART176V using FuGene6. After 30h, cells were treated with TCPOBOP (250nM) for 24h. Human primary heptocytes were kindly provided by Dr Stephen S. Ferguson (Life Technologies Corporation, Durham, NC, USA). Hepatocytes were treated with RIF (10µM) or CITCO (250nM) in William's E medium supplemented with 2mM l-glutamine, penicillin (100U/ml) and streptomycin (100µg/ml) for 24h.
Real-time PCR was performed using an ABI prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). Assays-on-Demand probes (Applied Biosystems) were used for PCR with the TaqMAN PCR Master Mix (Applied Biosystems). To measure the expression of human STS and CYP7A1 genes, SYBR Green PCR Master Mix (Applied Biosystems) was used with specific primers. The TaqMAN human β-actin probe (Applied Biosystems) was used as an internal control.
cDNAs for hHNF4α and hHNF3β in pcDNA3.1-V5-His-TOPO vector were in vitro translated using the TNT-coupled reticulocyte lysate system (Promega) according to the manufacture's instructions. Gel shift assays were performed as described previously (18). For competition or antibody supershift assays, unlabeled oligonucleotide, normal IgG, anti-HNF4α or anti-HNF3β antibodies was added 15min before adding radioactive oligonucleotides.
Recombinant GST and GST-fusion proteins of hPXR and hHNF4α were expressed in Escherichia coli BL21 (DE3) cells, and were purified through glutathione-Sepharose. A series of cDNAs for hPXR deletions and hHNF4α in pCR3 or pcDNA3.1-V5-His-TOPO vector were in vitro translated in the presence of [35S] methionine using a TNT-coupled reticulocyte lysate system (Promega) according to the manufacture's instructions. GST pull-down assays were carried out as described previously (19).
ChIP assays were performed using a ChIP assay kit (Millipore) according to the manufacture's instruction. Thirty hours after infection with adenovirus or reverse transfection, cells were treated with RIF in FBS-free MEM for an another 2 or 6h, and then cross-linked by adding formaldehyde [final 1% (v/v)] for 10min at room temperature. Cells were lysed in SDS lysis buffer containing protease inhibitor cocktail (Roche Applied Science) and sonicated to shear chromatin to lengths between 200 and 600bp. After pre-clearing by shaking with Protein A or G agarose/salmon sperm DNA, cell lysates were incubated with 3µg of antibodies or normal IgG at 4°C for 16h. After incubation with Protein A or G agarose/salmon sperm DNA, the immunocomplexes were collected by centrifugation. After de-crosslinking and protease-digestion, the immunoprecipitated DNA was purified by following the manufacture's instructions combined with the QIAquick PCR purification kit (Qiagen). The purified DNA was used as a template for semi-quantitative and real-time PCRs with specific primers.
3C assays were performed as previously described with minor modifications (20,21). Thirty hours after infection with adenovirus, Huh7 cells were treated with RIF in FBS-free MEM for another 6h. For 3C assays with Luc reporter constructs, trypsinized Huh7 cells were reverse transfected with each construct, using FuGene 6. Twenty-four hours after transfection, the culture medium was replaced with FBS-free MEM and the cells were incubated for another 2h. Cells were cross-linked by adding formaldehyde [final 1% (v/v)] for 10min at room temperature, and the reaction was stopped by adding glycine (final 125mM). After washing with ice-cold PBS, cells were incubated in a lysis buffer [10mM Tris, pH 7.5, 10mM NaCl, 0.2% (v/v) NP-40] and protease inhibitor cocktail on ice for 15min, followed by homogenization in a dounce homogenizer. After centrifugation, nuclei were collected as a pellet and suspended in restriction enzyme buffer 2 containing 0.3% (w/v) SDS. After incubation at 37°C for 1h, Triton X-100 was added to 1.8% (v/v) final concentration, followed by incubation for another 1h at 37°C. Aliquots of 1.5×106 nuclei were incubated with 500U of BstYI at 37°C for 16h. The enzyme reaction was terminated by adding SDS to 1.6% (v/v) and incubated for 20min at 65°C. The digestion efficiency was confirmed by gel electrophoresis on a 2.0%, agarose gel. An aliquot of digested chromatin was diluted with T4 DNA ligase buffer to 1% (v/v) final concentration Triton X-100 and 2.5ng/μl of DNA and incubated at 37°C for 1h. The DNAs were ligated by using 1600 cohesive end units of T4 ligase for 4h at 16°C followed by 30min at room temperature. Proteinase K, NaCl and EDTA were added to the ligation mixture to final concentrations of 40µg/ml, 0.2M and 1mM, respectively. These mixtures were incubated at 65°C to reverse cross-linking. After 16h incubation, mixtures were treated with RNase A at 37°C for 30min, and the DNA was purified by phenol extraction and ethanol precipitation. The purified DNAs were re-suspended in water at 50ng/µl, and subjected to semi-quantitative and real-time PCR of ligated and control fragments using specific pairs of primers.
Trypsinized cells were reverse transfected with pCR3/FLAGhPXR for 24h, and were subsequently treated with 0.1% (v/v) DMSO or RIF for another 2h. Then, cells were lysed in cold immunoprecipitation buffer [1% (v/v) Triton X-100, 150mM NaCl, 10mM Tris, pH 7.4, 1mM EDTA, 1mM EGTA, 0.2mM sodium ortho-vanadate, 0.2mM PMSF, 0.5% (v/v) NP-40 and 0.1% (v/v) DMSO or 20µM RIF] containing phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) at 4°C for 25min, and were centrifuged at 16000g for 15min to prepare whole cell lysates. For co-immunoprecipitation assays, FLAG-M2 agarose was incubated with cell lysates at 4°C for 2h, washed with immunoprecipitation buffer and subjected to Western blotting with anti-FLAG-M2 HRP-conjugated antibody or anti-V5 HRP-conjugated antibody.
All data are presented as mean±SD. Analysis was performed by using Student's t-test. A statistical probability of P<0.05 was considered significant.
Real-time PCR, performed using RNAs prepared from human primary hepatocytes treated for 24h with the human PXR activator RIF, determined that SULT1E1 mRNA levels decreased to approximately one-third of those in DMSO-treated hepatocytes after RIF treatment, while STS mRNA showed no change (Figure 1A). The CYP3A4 mRNA was greatly increased as expected. Repression of the SULT1E1 gene by RIF was also observed in human hepatocellular carcinoma Huh7 cells in which PXR was ectopically expressed through infection of an adenovirus. RIF treatment decreased the levels of SULT1E1 mRNA in a time-dependent manner in Huh7 cells infected with adeno-hPXR, but not with adeno-β-gal; the decrease began 6h after treatment and continued to an ~85% decrease after 16h (Figure 1B). Levels of STS mRNA, on the other hand, were maintained in Huh7 cells even after RIF treatment (Supplementary Figure S1A). These PXR-mediated expression patterns were conserved in HepG2 cells (Supplementary Figure S1B). Compared to the rapid increase in CYP3A4 mRNA, which began 2h after RIF treatment and peaked at the 6h mark, the gradual and slow decrease in SULT1E1 mRNA levels prompted us to examine whether or not protein synthesis was involved in this repression. Co-treatment with cycloheximide that inhibits translation did not block this decrease of SULT1E1 mRNA levels, thus indicating that protein synthesis is not required for PXR to repress transcription of the SULT1E1 gene (Figure 1C). Similarly, cycloheximide did not inhibit RIF induction of the CYP3A4 mRNA.
First, we found that the 5.3kb SULT1E1 promoter-Luc reporter was repressed ~50% by RIF treatment in PXR-expressing Huh7 cells (Figure 2A). Given the fact that the internal deletion (Δ−1.0/−0.8kb) abolished this repression (Figure 2A), further deletion analysis delineated the PXR-mediated repression activity to a distal 100bp composite enhancer sequence (−1000/−901bp) (Figure 2B). This enhancer sequence contains three direct repeat (DR) half sites that constitute the overlapping DR1 and DR2 motifs and two forkhead protein binding sites FHa and FHb. To determine the role of these half sites, they were mutated singularly or simultaneously within the context of the 5.3kb SULT1E1 promoter. Mutation of DRb (DRb-mt) decreased activity of the promoter and eliminated its ability to respond to PXR, thereby repressing activity of the reporter (Figure 2C). While single mutation of either DRa or DRc fully retained PXR-mediated repression of the promoter, double mutations of both DRa and DRc abolished this repression. Thus, these results indicate that the DRb plays a central role in the repression of the promoter activity. FHa and FHb sites were also mutated to examine their role in repression of activity of the 5.3kb SULT1E1 promoter; only the double mutation abolished the response of the promoter to PXR (Figure 2D). Taken together, these results lead us to conclude that it is the DRb site that plays the most critical role in the PXR-mediated repression of the the 5.3kb SULT1E1 promoter, although the two FH sites may also be involved in this repression.
Subsequent to the confirmation by gel shift assays that HNF4α and HNF3β bind to the DR and FH sites, respectively (Supplementary Figure S2), we employed siRNAs to knockdown HNF4α and HNF3β in order to examine their roles in regulating the SULT1E1 gene in Huh7 cells. Both knockdowns were found to decrease SULT1E1 mRNA levels (Figure 3A). Knockdown of HNF4α was the most effective in decreasing SULT1E1 mRNA, and reduced the mRNA levels by 90%. On the other hand, knockdown of HNF3β only reduced levels of SULT1E1 mRNA by ~40%. The HNF3β knockdown also resulted in a small reduction of HNF4α, which is consistent with the fact that HNF3β is one of the transcription factors that activates the HNF4α gene (22). To examine interactions of the 100bp enhancer with HNF4α and HNF3β, ChIP assays were performed for the SULT1E1 gene in Huh7 cells. Both HNF4α and HNF3β bound to the enhancer region of the SULT1E1 promoter, but not to the proximal promoter region (Figure 3B). These results indicate that HNF4α may play the most critical role in enhancer activation, although HNF3β is also involved in this activation.
Utilizing deletion promoters, the regions required for repression of activity by PXR were localized in the SULT1E1 promoter. The activity of a −1.1kb promoter was repressed by PXR, while a −1.1kb-Δ promoter, which places the enhancer in front of a 105bp proximal promoter via an internal deletion, greatly enhanced the basal transcription activity and fully retained the repression of activity by PXR (Figure 4). However, the 100bp enhancer repressed promoter activity when it was placed directly in the front of the TK promoter. Thus, the repressive activity was minimized to the 100bp enhancer and the −105bp proximal promoters (Supplementary Figure S3). Noticing that the −1.1kb-Δ promoter exhibited higher activity than the −1.1kb promoter, we hypothesized that the SULT1E1 promoter adopts a looped structure in its active conformation. Utilizing the restriction enzyme BstYI to digest the SULT1E1 gene, 3C assays were designed to detect a 135bp DNA fragment by PCR amplification upon ligation of the BstYI sites that are located −1832bp and +39bp from the transcriptional start site (Figure 5A). Accordingly, this 3C assay demonstrated that the promoter was looped in its active conformation (Figure 5B). The same 3C assays were performed on the SULT1E1 gene in PXR-expressing Huh7 cells after RIF treatment; amplification of a 135bp DNA fragment was detected by both semi-quantitative and real-time PCR. The degree of amplification was strongly attenuated in the presence of PXR after RIF treatment (Figure 5C). Subsequently, ChIP assays were employed on the SULT1E1 gene using an antibody that detects acetylated histone 3 at lysine 9 and lysine 14 (H3K9K14ac); acetylation of histone 3 is known to facilitate activation of gene transcription. H3K9K14ac was reduced on both the enhancer and proximal promoter regions of the SULT1E1 gene in PXR-expressing Huh7 cells after RIF treatment (Figure 6A and B). Thus, the PXR-dependent chromatin structure disruption as observed in our 3C assays correlated with this deacetylation of histone 3 on the SULT1E1 gene.
Next, ChIP assays were performed to determine the interactions of HNF4α, HNF3β and PXR with the SULT1E1 promoter. Binding of both HNF4α and HNF3β to the enhancer was attenuated after RIF treatment in PXR-expressing Huh7 cells; no such attenuation was observed with control β-gal-expressing Huh7 cells (Figure 7A and B). HNF4α and HNF3β were not detected on the proximal promoter even after RIF treatment under our assay conditions. There was no PXR binding to either the enhancer or to the proximal promoter before or after RIF treatment (Figure 7A). Thus, PXR appeared to decrease the binding of HNF4α and HNF3β to the enhancer, repressing SULT1E1 promoter activity. To further distinguish the roles of HNF4α and HNF3β, plasmid-based 3C assays were developed in which the 5.3kb SULT1E1 promoter plasmid was transfected into Huh7 cells and used as template. BstY1 digestion was designed to produce a 111bp DNA fragment when the promoter adopted a loop structure (Figure 8A and B). Within the context of the 5.3kb SULT1E1 promoter, either DRb or both FH sites were mutated and these mutated promoters were subjected to 3C assays. Mutation of the DRb disrupted the loop structure of the promoter by ~60%, whereas only <10% was decreased after mutation of the FH sites (Figure 8C). This weak contribution by FH sites to the loop structure was consistent with the less effect of HNF3β knockdown on the expression of SULT1E1 gene (Figure 3A).
Co-immunoprecipitation assays demonstrated that PXR formed a complex with HNF4α in Huh7 cells, and that formation of this complex formation was increased following RIF treatment (Figure 9A). No such increase was observed with HNF3β. These results, therefore, lead us to conclude that it is the HNF4α that plays the primary role in adopting the SULT1E1 promoter in the active loop formation, and that PXR targets HNF4α to disrupt this loop.
Here, we have identified the upstream enhancer of the SULT1E1 promoter that plays a crucial role in regulating the activity of SULT1E1 transcription. HNF4α binds to the enhancer and acts as a mediator that enables the SULT1E1 promoter to form an active chromatin structure by placing the enhancer close to the proximal promoter, thereby determining the basal rate of transcription. Upon activation by RIF, PXR, through its interactions with HNF4α, prevents the promoter from forming the active chromatin structure, thereby repressing transcription of the SULT1E1 gene. PXR was previously reported to repress the expression of the G6Pase and CYP7A1 genes through its interactions with HNF4α and PGC1α (4,5,19). However, PGC1α does not appear to be involved in the repression of the SULT1E1 gene, since the transcription of the SULT1E1 gene was not affected by siRNA knockdown of PGC1α in Huh7 cells (Supplementary Figure S4). The chromatin structure-mediated repression mechanism for the SULT1E1 gene differs from the previous PGC1α-dependent gene repression by PXR. FOXO1, HNF3β and CREB were also suggested to be PXR targets for the protein–protein interactions, which repress the expression of the G6Pase, CPT1A and HMGCS2 genes, the molecular mechanism for which remains unclear (3,4,23). Thus, SULT1E1 appears to be the first gene whose repression by PXR is now understood at the level of chromatin structure.
PXR interacted with HNF4α in response to RIF activation, suggesting that this interaction can be a critical regulatory step within the underlying mechanism, by which PXR represses the expression of the SULT1E1 gene. Gel Shift assays indicated that PXR does not directly bind to any DNA sequence within the 100bp enhancer (data not shown). Therefore, if in fact PXR interacted with the 100bp enhancer, this interaction would have occurred via protein–protein interactions. However, our ChIP assays did not confirm that PXR interacted with either the enhancer or the proximal promoter of the SULT1E1 gene in RIF-treated Huh7 cells. Several reasons for this can be speculated, one of which is that the interaction of PXR with HNF4α on the SULT1E1 gene is transient, so that ability of ChIP assay to detect this transient interaction may be limited (Figure 9B). Although GST pull-down assays determined that PXR and HNF4α bind through their DNA binding domains, gel shift assays found that PXR does not inhibit binding of HNF4α to the overlapping DR1 and DR2 motifs within the 100bp enhancer (Supplementary Figures S5 and S6). Therefore, PXR does not appear to just simply prevent the binding of HNF4α. In this respect, HNF4α activity can be regulated by post-translational modifications such as acetylation and phosphorylation within its DNA binding domain and hinge region (24–26). PXR may also utilize these phosphatases and deacetylases to repress the HNF4α-mediated activation of the SULT1E1 gene, which presents an interesting possibility for future investigations to decipher the details of this repression mechanism.
The liver is an estrogen-targeted organ, although whether or not hepatic estrogen metabolism critically regulates liver function in an autocrine and or paracrine fashion remains to be further explored. Estrogen receptor α (ERα) KO mice developed hepatic insulin resistance by up-regulating lipogenic enzymes in the liver (27). Protein S is a major risk factor for developing thrombosis and the liver is the site of its production. ERα activation is known to repress the protein S gene in mouse liver (28). Estrogens regulate hepatic bile acid synthesis and a sustained activation of ERα by 17α-ethynylestradiol produces hepatotoxicity in mice (29). Utilizing Pxr−/− mice, we recently determined that PXR represses the hepatic Sult1e1 gene during fasting (data not shown). CAR, a closely related nuclear xenobiotic-activated receptor, was reported to activate the SULT1E1 gene in mouse liver (11). In human primary hepatocytes, on the other hand, drug activation of CAR resulted in repression of the SULT1E1 gene (30) (Supplementary Figure S7). Moreover, similar to PXR, CAR repressed the 5.3kb SULT1E1 promoter and the expression of the endogenous SULT1E1 gene in Huh7 cells (Supplementary Figure S8). In addition to SULT1E1, PXR and CAR can also regulate the expression of enzymes that metabolize estrogens, such as CYP3A4, following activation by an overlapping group of xenobiotics and therapeutics. Thus, PXR and CAR may co-operate in regulating hepatic levels of active estrogens, thereby affecting the physiology and/or pathophysiology of human liver.
Supplementary Data are available at NAR Online.
Intramutal Research Program of the National Institutes of Health and National Institute of Environmental Health Sciences: Z01ES1005-01. Funding for open access charge: NIH.
Conflict of interest statement. None declared.
We thank the NIEHS sequence core for excellent assistance. We also thank Mr Rick Moore at NIEHS for technical assistance.