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CYP2Cs and CYP3A4 sub family of enzymes of the Cytochrome P-450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR-HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors.
Cytochrome P450 enzymes constitute a super family of hemoproteins found throughout the animal kingdom . CYP2C9, the major member of the CYP2C subfamily in human liver, metabolizes more than 16% of clinically used drugs, and forms physiologically active metabolites from the endogenous substrate arachidonic acid [2,3]. Studies in primary hepatocytes have shown that, CYP2C9 mRNA and protein to be up regulated by numerous drugs such as rifampicin, hyperforin (the active ingredient in St. John’s Wort), phenobarbital and taxol [4–8]. Furthermore, clinical studies have confirmed that the clearance of CYP2C9 substrates is increased in vivo after the administration of drugs [9–12].
Studies have shown that the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) both bind to the same responsive elements (RE’s) at −2998 (DR5) and −1839 (DR4) to mediate the transcriptional activation of the CYP2C9 gene by various drugs [13–15]; however, the proximal site at −1839 appears to be the more important site. PXR belongs to the NR1I2 nuclear family of receptors [16,17], which are activated by a wide range of structurally unrelated compounds [18–21]. PXR preferentially binds many xenobiotic ligands such as rifampicin, taxol, and hyperforin [22,23]. Ligand dependent PXR activation up regulates many of the phase I and II, drug metabolism genes and I and genes involved in transport and clearance pathways including bile acid homeostasis . There is considerable cross talk between PXR and CAR for similar sets of responsive elements in the DNA of various promoters [23,25,26]. Hetero-dimerization of PXR and CAR with RXR (retinoid X receptor) facilitates binding DR-3, DR-4 or DR-5 sites in the promoter region of various genes [18,27–31].
CYP2C9 and CYP3A4, like many other CYP genes, are preferentially expressed in the liver and developmentally regulated by hepatic enriched transcriptional factors such as HNF4α (hepatic nuclear factor 4α) [32–34]. HNF4α activates the transcription of target genes through recognition of a direct repeat (DR1) motif and recruitment of the chromatin remodeling system [35,36]. Three proximal DR-1 sites have been reported in the CYP2C9 gene which bind HNF4α and increase transcription in response to HNF4α . Studies in our laboratory have reported cross talk between the CAR and HNF4α binding sites in the CYP2C9 promoter . HNF4α and CAR/or PXR both synergistically activate the CYP2C9 promoter in HepG2 cells.
Our present study addresses the mechanism of the cross talk between PXR and HNF4α. GST pull-downs show that NCOA6 interacts with PXR and HNF4α in the presence and absence of the PXR ligand rifampicin. Mapping the interacting domains, we have shown that the first LXXLL motif is essential for PXR interaction, while both the first and second LXXLL motifs were involved in the interaction with HNF4α. We have confirmed the interaction in vivo with mammalian two-hybrid assay. Ectopic expression of NCOA6 modestly arguments the activation of the CYP2C9 and CYP3A4 promoters by PXR and HNF4α individually and the synergistic activation by the combination of these two receptors. However, silencing endogenous NCOA6 abolished the synergistic activation of the CYP2C9 but not that of the CYP3A4 promoter. Similarly, NCOA6 differentially regulated the increase in CYP2C9 and CYP3A4 mRNA after over expression of PXR and HNF4Α individually or in combination. Finally, chromatin immunoprecipitation (ChIP) analysis showed that PXR and HNF4α were recruited to their respective binding sites on the CYP2C9 promoter and NCOA6 bound to both sites. These studies are consistent with the hypothesis that NCOA6 forms a bridge between the PXR and HNF4α binding sites in the CYP2C9 promoter providing the platform for the recruitment of other co activators to facilitate the cross talk between these distal sites.
HepG2 cells were maintained in the Eagle’s minimal essential medium supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C under 5% CO2. All transient transfections were carried out as described in Lipofectamine 2000 protocol (Invitrogen, CA). Briefly, 0.2 µg of CYP2C9 or CYP3A4 luciferase constructs, 0.1µg of each receptor construct(s), and 0.2 µg of co activator construct with 0.02 µg of pRL-TK vector as internal control, pcDNA 3.1 as the empty vector to make the total amount of DNA transfected to 0.8 µg were combined in 50 µL OPTI-MEM and mixed with transfection reagent as suggested. Twenty-four hours later, medium was replaced, and drugs were added at the appropriate concentrations (0.1% of DMSO or 10 µM rifampicin). Ligands were incubated with the HepG2 cells for 24 h and assayed for promoter activity using Promega’s dual luciferase assay kit (Promega, Madison, WI). Firefly luciferase readings were normalized with renilla readings to calculate promoter activity.
PXR, HNF4α, NCOA6, Med1, PGC-1a and SRC-1 were cloned in pcDNA3.1 by PCR amplification. GST-PXR and GST-HNF4α were cloned in pGEX-4T1. NCOA6 domains, NCOA6 I (1–353 aa), NCOA6 II (338–673 aa), NCOA6 III (648–998 aa containing 1st LXXLL motif at 851 aa), NCOA6 IV (986–1327 aa), NCOA6 V (1292–1641 aa coding for 2nd LXXLL motif at 1491 aa) and NCOA6 VI (1625–2065aa) were cloned in pGEX-4T1 in-frame of GST and pACT for expression in two hybrid assay. For transient transfection assay of CYP2C9 promoter expression, CYP2C9-1.9Kb/pGL3 construct has been previously described (Chen et al 2005). CYP3A4 promoter construct was a kind gift (Dr. Stephen Kliewer, Southwestern University Texas Medical School, Dallas, TX). Full-length PXR and HNF4α were cloned into pShuttle-CMV vector of AdEasy™ XL Adenoviral Vector system (Stratagene, CA) at the EcoRV site. PmeI linearized shuttle vectors were electroporated into BJ5183-AD1 cells containing the Ad Easy vector backbone for homologous recombination. Positive recombinant Adeno plasmids were selected on kanamycin and confirmed with restriction digestion. The recombinant plasmids were digested with Pac I and transfected into AD-293 cells for replication and packaging into virus particles. Virus particles were purified on continuous cesium chloride, dialyzed and stored in Tris buffered sucrose. pShuttle-CMV-lacZ supplied as control was used for making Ad-lacZ virus. shRNA were designed to silence the NCOA6 mRNA coding sequence were described earlier . Double stranded shRNA oligos were designed for pRNAT-H1.1/Adeno (SD-1219) with H1 promoter and cGFP as the marker. The final NC-III shRNA target sequence was used to design the scrambled shRNA 5’-CACTGAAGTATACCAAGAGCA-3’. Adenoviruses expressing each shRNA were prepared as described before. HepG2 cells were routinely infected with 2.5 × 109 virus particles (VP).
Full-length recombinant proteins of PXR and HNF4α were expressed as GST fusion proteins in E.coli BL21 (DE3). Briefly, sequenced verified pGEX4T-PXR/HNF4α constructs were transformed in E.coli BL21 and initially screened for protein expression. Liquid cultures grown up to A600 of 0.4 were induced with 1 mM IPTG at room temperature for 3h and the bacteria were collected. The cells were lysed by three freeze thaw cycles and suspended in TEDG (Tris 50mM pH 7.4, 2 mM EDTA, 2 mM DTT and 10% glycerol) buffer containing 0.42 M NaCl, 1% Triton X -100 and complete protease inhibitors. The lysate was centrifuged at 100,000 × g for 45 min and the supernatant was used for immobilizing GST fusion proteins on GSH-sepharose matrix. Immobilized proteins were washed 3–4 times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride) and analyzed by SDS-PAGE for purity and quantification of the fusion proteins. GST pull-down assays were performed by incubating 5 µL of 35S methionine labeled proteins in a 500 µL of NETN buffer containing 1mg/ml fatty acid free BSA in the presence and absence of the PXR ligand rifampicin. The bound proteins were washed 3X with NETN buffer and were heat denatured in SDS sample buffer. These proteins were separated on 4–20% gradient gels and after fixing and amplifying the signal; the gels were dried and autoradiographed.
To map the interaction domains between NCOA6 and HNF4!, or NCOA6 and PXR, the ligand-binding domain of HNF4! and PXR were fused to the GAL4 DBD in a pBind vector individually. NCOA6 domains from the previous GST fusion constructs were transferred to pACT vector individually. The promoter luciferase reporter 9XGAL4 UAS was employed to assay the interaction in HepG2 cells along with the pBind construct and pACT constructs as shown. Promega’s Dual-Glo System was used to measure the luciferase activity.
Total RNA was extracted using RNeasy mini prep system (QIAGEN, Valencia, CA) following the manufacturer's procedure. RT-PCR analysis was performed in two steps by initial reaction with Superscript II (Invitrogen) reverse transcriptase. PCR with Taqman® Universal PCR Master Mix (Applied Biosystems) was then performed with gene-specific primers using relative quantification methods. For the RT reaction, 200 ηg of total RNA was combined with 2 µl (40 units) of RNase inhibitor (PerkinElmer Life and Analytical Sciences, Boston, MA), 1× First Strand Buffer (Invitrogen), 10 mM dithiothreitol, 0.5 mM dATP, dTTP, dGTP, and dCTP, and 1 µl (200 units) of Superscript II to a total volume of 20 µl, incubated at 42°C for 50 min and then inactivated at 70°C for 15 min, and stored at −20°C or immediately used in PCR analysis. PCR was performed on an Applied Biosystems 7900HT using relative quantification 2−ΔΔCT method with taqman probes for CYP2C9 (Cat# HS00426397_m1. lot# 465955), with TBP as the internal control (4333769F, lot# 0706051).
Five plates (15 cm) of 90% confluent HepG2 cells were infected with 2.5 × 1011 VP for 48 h each with adenovirus expressing, LacZ, PXR, HNF4α, PXR-HNF4α and PXR-HNF4α with shRNA for NCOA6 individually. A similar set of HepG2 cells were treated with 10µM rifampicin. After 48h, the cells were cross-linked with 1% formaldehyde directly in the media, and cross-linking was stopped by the addition of 0.125 M glycine. The cells were washed in ice cold PBS and lysed in hypotonic solution (5mM Pipes). The nuclei were harvested after 30 min, washed and solubilized in Buffer A containing 50mM Tris-Cl, pH 8.0, 1% SDS, 5 mM EDTA, 5 mM EGTA, 0.5 mM PMSF and complete protease inhibitor mix (Roche Molecular Biochemicals). The homogenate was sonicated 6–7 times on ice at 40% setting (Branson sonicator) to shear the chromosomal DNA to an average length of <500 bp. The insoluble material was removed by high-speed centrifugation and chromatin extracts were stored at −80°C. The soluble chromatin was diluted 5 fold with Buffer B containing 20 mM Tris-Cl pH 8.0, 1% Triton X-100, 1.2 mM EDTA and 150 mM NaCl. The chromatin was diluted and precleared by incubating for 3 h with 5µL of pre-immune serum coupled to protein-A agarose beads saturated with BSA (1 mg/mL) and salmon sperm DNA (0.4 mg/mL). Precleared chromatin was incubated with 2 µg of individual antibodies overnight, the complexes were precipitated with 50 µL of 50% protein A agarose beads. The complexes were washed 2X with 1 mL buffer C (50 mM Tris-Cl pH 8.0, 2 mM EDTA, 0.2% sarkosyl), and 4X with 1 mL buffer D (100 mM Tris-Cl pH 9.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid) sequentially. Immune complexes were eluted from the protein A beads by vortexing in 150 µL of Buffer E (50mM sodium bicarbonate, 1% SDS) for 15 min twice . The eluants were pooled and adding 5 M NaCl to a final concentration of 200 mM reversed the protein-DNA cross-linking. RNA was removed with the addition of 10 µg of DNase free RnaseA followed by incubation at 65°C for 4 h. Genomic DNA from the immune complexes was EtOH precipitated and the proteins in the complex were digested with 15 µg of Proteinase K. Finally, the DNA binding proteins bound cognate cis acting elements were purified with phenol-choloroform-isoamyl alcohol and further on qiagen columns. Similarly purified DNA fragments from the chromatin extracts (input) were used as control for PCR reactions. The primers used for amplification of the human CYP2C9 promoter are 5’-TAAAGACAGCAACCGAGC-3’ and 5’-TACAATGATTCAGGATTTCG-3’ spanning for the proximal CYP2C9 PXR response element and 5’-ATATACAAGGCATAGAATATGGCC-3’ and 5’-GACCAATCACCTAGGTCCAC-3’ spanning the CYP2C9 HNF4α response elements.
We have previously shown that NCOA6 interacts with CAR and HNF4α and silencing NCOA6 interferes with the synergistic activation of the CYP2C9 promoter by HNF4α and CAR . In this paper, we tested if NCOA6 would also interacts with PXR? A partial fragment of NCOA6 coding for the first LXXLL motif was expressed as a fusion protein of GST, which was immobilized on GSH sepharose beads. Full-length hPXR and HNF4α were labeled with 35S methionine and allowed to interact with the immobilized GST-NCOA6 fragment. Both PXR and HNF4α interacted strongly with the first LXXLL motif of NCOA6, and the PXR ligand rifampicin had no significant effect on these interactions (Fig. 1a and b). Reciprocally, we have immobilized full length human PXR (hPXR) and hHNF4α expressed as GST fusion proteins, allowed them to interact with full length NCOA6 and known co activators such as Med1, GRIP-1, SRC-1 and PGC-1α in the presence and absence of rifampicin. NCOA6 interacts with PXR robustly, and the PXR ligand rifampicin has little effect on this interaction. Med1, the anchoring protein of the mediator complex, GRIP-1, SRC-1 are part of the CBP bound HAT containing enzyme complex and PGC-1α is a co activator implicated in energy regulation. These established co activators are known to interact with most nuclear receptors and were tested for their ability to interact with PXR and HNF4α (Fig. 1c). Although rifampicin is not a ligand for HNF4α, the presence of rifampicin appeared to enhance the binding of co activators to HNF4α suggesting that rifampicin might alter fatty acid binding to the HNF4α ligand-binding pocket.
To define the interacting domain(s) of NCOA6, GST-NCOA6 domains were allowed to interact with 35S-methionine labeled PXR. GST and GST-NCOA6 fusion proteins are shown schematically in Fig. 2a. GST-NCOA6-III domain (648–998aa contains the first LXXLL at 851aa) interacted significantly with PXR (Fig. 2b) in a ligand independent manner. No other NCOA6 domain was found to bind PXR suggesting that only the first LXXLL motif was involved in the interaction. Previously, the first LXXLL motif of NCOA6 was shown to interact with PPAR nuclear receptors (α and γ) TRα and ERα . Taken together, these data suggest that there is only one functional interaction domain. When the interacting domains of NCOA6 and HNF4α were similarly mapped with 35S-methionine-HNF4α , the GST-NCOA6-III domain strongly retained the radio labeled HNF4α, but there was also a moderate interaction between the GST-NCOA6-V domain (containing the second LXXLL motif) and HNF4α, suggesting a possible additional role for this LXXLL motif. This second LXXLL motif of NCOA6 was shown to be involved in the interaction with LXR (another liver specific receptor) in regulating lipogenesis and cholesterol/bile acid homeostasis in the liver and interaction with ERα with no significant physiological function .
To assess the strength and specificity of the interaction between NCOA6 and the nuclear receptors PXR and HNF4α, we employed a mammalian two-hybrid assay. The six partial fragments shown in Fig. 2a spanning the entire coding region, were cloned in pACT vector to express each as a fusion protein downstream of the VP-16 activating domain. The ligand binding domain of HNF4α and PXR were cloned individually in pBind vector to express each as a fusion protein of the GAL4 DNA binding protein. All the constructs were individually tested for activation with the promoter construct pSG9. An interaction assay with the HNF4α ligand binding domain and the NCOA6 fragments showed that the first LXXLL motif was essential for the interaction and there was a small interaction with the second LXXLL motif too (Fig. 3a). On co expression of the ligand-binding domain of PXR with the activation domain of VP-16 fused to NCOA6 fragments, again the domain III that codes for the first LXXLL motif interacted with the ligand-binding domain of PXR, which could be further, boosted by the presence of rifampicin (Fig. 3b). There was small but significant interaction with the second LXXLL motif (domain V). Thus, although the second LXXLL motif showed a small interaction with PXR in vivo and with HNF4α in vitro, it did not show significant enhancement of ligand dependent transcription activation in the two-hybrid assay for the PXR.
PXR transactivates the CYP2C9 and CYP3A4 promoters and the activation is enhanced by the presence of 10 µM rifampicin as the ligand. Expression of NCOA6 with PXR has no significant effect on PXR mediated transactivation of either the CYP2C9 or CYP3A4 promoters in the presence of rifampicin (Fig. 4). Ectopic HNF4α also activated CYP2C9 promoter activity but had no effect on CYP3A4 promoter activation. When NCOA6 was ectopically co expressed with HNF4α there was a modest enhancement of activity of CYP2C9 and CYP3A4 promoter activity, which was augmented further in the presence of rifampicin (Fig. 4). But when PXR and HNF4α were co expressed with the CYP2C9 or CYP3A4 reporter constructs, there was a synergistic increase in CYP2C9 and CPY3A4 promoter activity specifically in the presence of rifampicin (Fig. 4). In the absence of PXR ligand rifampicin, the synergistic activation is not seen, suggesting that the ligand is required for the PXR activation, which could assist in correct refolding of the AF2 domain on PXR facilitating the recruitment of co activators, which in turn bind to HNF4α. Addition of ectopic NCOA6 boosted this activation several fold for both the promoters (CYP3A4 and CYP2C9) only upon the synergistic activation. In conclusion, there is a synergistic activation of CYP2C9 and CYP3A4 promoter expression by the nuclear receptors PXR and HNF4α particularly in the presence of the PXR ligand, rifampicin. The synergism was moderately augmented by NCOA6 consistent with a possible role for the co activator in the regulation of CYP2C9 and CYP3A4 by PXR and HNF4α.
We have conclusively demonstrated that NCOA6 is a bonafide co activator for HNF4α . To test its transcriptional role in the activation of CYP2C9 by PXR and HNF4α, we expressed small interfering RNA (shRNA) directed against NCOA6 in HepG2 cells using an adenoviral system to silence NCOA6 expression. Among five targets, NC-III and NC IV reduced the expression of NCOA6 mRNA levels by 90% and 75% respectively as quantified by qPCR . To test whether silencing of NCOA6 affects the ligand dependent synergistic activation of the CYP2C9 promoter by PXR and HNF4α, the CYP2C9-luc promoter construct and PXR, HNF4α or PXR-HNF4α were transiently transfected into HepG2 cells, and 24 h later the cells were infected with adenovirus expressing scrambled or shRNA for NCOA6(NC-III). The synergistic activation of the CYP2C9 promoter by PXR-HNF4α could be dramatically repressed by the expression of shRNA for NCOA6 in the presence of a ligand but not in absence of ligand rifampicin (Fig. 5a). However, silencing NCOA6 did not have any effect on the synergistic activation of the CYP3A4 promoter (Fig. 5b).
HepG2 cells express CYP2C enzymes poorly, possibly due to low expression of factors such as CA, PXR, PGC-1 and/or SRC-1. We examined the effect of silencing of NCOA6 on CYP2C9 mRNA in HepG2 cells. The shRNA for NCOA6, down regulated the constitutive levels of CYP2C9 mRNA by 75% compared to the scrambled shRNA controls. (Fig. 6a) In contrast, ectopic over expression of PXR or HNF4α induced substantial amounts of CYP2C9 mRNA levels, but NCOA6 shRNA failed to represses this induction, suggesting that NCOA6 may not play a direct role in the PXR or HNF4α mediated up regulation of the transcription of CYP2C9 (Fig 6b and 6c). Silencing NCOA6 with shRNA decreases the NCOA6 protein levels as seen in the western blot compared to lacZ or scrambled virus. However RNA Pol II as a control does not change its expression when NCOA6 protein levels were diminished (Fig. 6d), suggesting that shNCOA6 is specifically acting towards down regulating the expression of NCOA6 alone. On the other hand, we have shown above that the down regulation of NCOA6 levels inhibits the synergistic activation of CYP2C9 promoter activity by the ectopic expression of PXR and HNF4α (Fig 5 a). To translate these data to gene expression, we infected AdPXR and AdHNF4α individually in HepG2 cells and simultaneously down regulated NCOA6 levels with shRNA for NCOA6. Expression of PXR and HNF4α produces a synergistic 50-fold induction of CYP2C9 mRNA in HepG2 cells in the presence of rifampicin compared to a less then 5-fold induction by HNF4α or PXR in the absence of rifampicin. The synergistic induction by ectopic expression of HNF4α and PXR on CYP2C9 mRNA in HepG2 cells was essentially abolished by down regulating NCOA6 with adenoviral shRNA (Fig. 7a). In contrast there was no synergistic induction of CYP3A4 mRNA by ectopic expression of PXR and HNF4α and silencing NCOA6 had no effect on its induction (Fig. 7b). Taken together, the transactivation and mRNA induction results strongly indicate that NCOA6 may act as a bridging partner between PXR and HNF4α resulting in a synergistic up regulation of CYP2C9 gene expression but not for CYP3A4 gene expression.
ChIP assays demonstrated that PXR is recruited strongly to the PXR binding site on the promoter region of CYP2C9 in chromatin extracts prepared from HepG2 cells after ectopic expression of PXR and moderately after ectopic expression of PXR-HNF4α with or without infection with shRNA for NCOA6. As expected, HNF4α was recruited to its binding sites on the promoter in all the chromatin extracts from cells over expressing HNF4α, PXR-HNF4α and shRNA for NCOA6 did not affect this binding. A specific antibody to NCOA6 was able to immunoprecipitate the PXR and the HNF4α binding sites of CYP2C9 in chromatin extracts from cells ectopically expressing PXR and PXR-HNF4α. Silencing NCOA6 prevented immunoprecipitation of both the PXR and HNF4α binding sites of the CYP2C9 promoter by antibody to NCOA6 (Fig. 8). Thus ChIP assays show that PXR and HNF4α can bind to their respective binding sites on the CYP2C9 promoter in vivo and NCOA6 binds to both sites supporting the hypothesis that the NCOA6 may be necessary for formation of a bridge between the HNF4α and PXR sites and form the basis for the recruitment of other co activators involved in the massive induction of CYP2C9 gene expression by ectopic over expression of both PXR and HNF4α.
Hepatic drug metabolism is one of the mechanisms whereby organisms protect themselves from exposure to environmental chemicals and either activates or deactivates [34,37] therapeutic drugs. CYP2C9 is an important drug-metabolizing enzyme which metabolizes ~16% of all clinical drugs as well as some endogenous compounds. CYP2C9 is up regulated by drugs and other xenobiotics primarily via two xenobiotic sensing receptors, CAR (NR1I3) and PXR (NR1I2). PXR is one of the most important xenobiotic sensing receptors since it is promiscuous, interacting with a wide array of ligands including clinical drugs such as rifampicin, paclitaxol, and dietary supplements such as St. John’s Wort. PXR regulates the response of a variety of CYP enzymes, drug transporters, and phase II drug metabolizing enzymes in response to environmental stimuli. Many of the CYP enzymes and phase II enzymes are found in high concentrations in liver, where HNF4α, a hepatic enriched factor, regulates both their constitutive expression and enhances their response to the xenobiotic sensing receptors CAR and PXR [41–43]. In fact, for a number of these enzymes such as CYP2C9 and CYP3A4, the receptors HNF4α and CAR have been reported to synergistically activate their promoter activity in cell lines, and studies in primary hepatocytes [34,44] and HNF4α knockout mice have confirmed that HNF4α plays a central role in regulating the response of drug metabolizing enzymes to PXR and CAR .
It has become apparent that cross talk with other transcription factors, coactivators and growth signals is necessary to explain the interplay between different receptors [45,46]. In these and recent studies , we performed yeast two-hybrid screens to identify receptors/and or co activators which might explain the interplay between these receptors. One of the co activators identified in a yeast two-hybrid screen using HNF4α as bait was the co activator NCOA6 which was subsequently shown to interact with the receptor CAR using protein-protein interaction techniques principally through the first LXXLL motif with some interaction through the second LXXLL motif . The present study shows that the xenoreceptor PXR also interacts with NCOA6 through the first LXXLL motif. Mammalian two hybrid assays further confirm that NCOA6 interacts with PXR and HNF4α principally through its first LXXLL motif although a smaller interaction is seen with the second LXXLL motif.
Exogenous NCOA6 has little effect on CYP2C9 or CYP3A4 promoter activity in the presence or absence of PXR, but increases HNF4α activation and the synergistic activation by PXR and HNF4α particularly in the presence of the PXR ligand rifampicin. Silencing NCOA6 decreased the synergistic activation of the CYP2C9 promoter by PXR and HNF4α, but surprisingly did not affect that of the CYP3A4 promoter. Parallel effects were seen on CYP2C9 and CYP3A4 mRNA. shNCOA6 preferentially affected the synergistic increase in CYP2C9 mRNA by HNF4α and PXR but had no effect on CYP3A4 mRNA induction.
There are many examples of crosstalk between CAR or PXR and HNF4α. Cross-talk between PXR and HNF4α has been reported for the human sulfotransferase SULT2A1 whereby this enzyme is up regulated by HNF4α and down regulated by PXR . Rifampicin induction of CYP3A4 has been proposed to involve cross talk with HNF4α and PXR via various co activators such as PGC-1α, SRC-1, and the small heterodimer partner (SHP) may compete with HNF4α and SRC-1 for PXR . The observed differential regulation of CYP2C9 and CYP3A4 could be due the fact that at the promoter level, the constructs of CYP2C9 and CYP3A4 are of similar length and the distance between the PXR binding site and the HNF4a binding site is about 1.6 kbp, which could be a possible explanation for the synergistic activation of the promoter constructs. But at the chromatin level, though CYP2C9 is about 1.6 kbp, the distance between the binding sites is about 8 kbp for CYP3A4. This long distance between the binding sites could prevent the proper loop formation required for the synergistic activation. Our data with mRNA and silencing of NCOA6 strongly supports this hypothesis. Negishi and coworkers  reported that the signal molecule EGR1 (early growth factor) binds to the proximal promoter of CYP2B6 and coordinates interaction with a nearby HNF4α site which cross-talks with a CAR binding at a distal enhancer site causing synergistic induction by drugs. A later paper from the same group provided evidence that EGR1 is involved in a loop formation between the distal enhancer and the proximal sites . Similarly, our studies are consistent with the hypothesis that NCOA6 may assist in loop formation between the distal PXR sites and the proximal HNF4α sites of the CYP2C9 promoter as depicted in the graphical abstract.
This study was supported by the Intramural Research Program of NIH, National Institute of Environmental Health Sciences, NIH intramural project number Z01ES02124.
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Conflicts of Interest
The authors declare that they have no conflict of interest.