Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease. However, mechanism(s) of this down-regulation have not been fully elucidated. As the pregnane X receptor (PXR) is involved in inducing many of these hepatic proteins, it is possible that PXR is also involved in their down-regulation during inflammation. Thus, we compared the effect of inflammation on hepatic gene regulation in wild-type (PXR+/+) versus PXR-null (PXR-/-) mice. Treatment of PXR+/+ but not PXR-/- mice with the PXR activators 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN) or 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl] estra-4,9-dien-3-one (RU486) resulted in increased mRNA levels of bsep, mdr1a, mrp2, mrp3, oatp2, and cyp3a11, indicating involvement of PXR in their regulation. Significantly lower mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were found in endotoxin-treated PXR+/+ mice. In endotoxin-treated PXR-/- mice, the extent of mrp2 suppression was significantly diminished. Changes in MRP2 expression were supported by Western blot analysis. Although interleukin (IL)-6 imposed significant decreases in the expression of bsep, mrp2, and cyp3a11 in PXR+/+ mice, this was not observed in PXR-/- mice. Of note, significantly lower levels of PXR mRNA and protein were detected in endotoxin- and IL-6-treated PXR+/+ mice. In addition, endotoxin and IL-6 were also able to suppress PCN-mediated induction of bsep, mrp2, cyp3a11, and PXR. Taken together, our results suggest that PXR plays a role in the down-regulation of several hepatic proteins during inflammation.
Endotoxin-induced sepsis, viral infections, and other inflammatory conditions are a relatively frequent cause of intrahepatic cholestasis in patients (Trauner et al., 1999). Disruptions in the hepatic accumulation and excretion of bile salts and acids occur due to down-regulation of both basolateral uptake [Na+ taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptide 2 (OATP2)] and canalicular efflux [bile salt export pump (BSEP), multidrug resistance associated protein (MRP2), P-glycoprotein, multidrug resistance (MDR1)] transport systems. The molecular mechanisms involved in this down-regulation have not been fully elucidated. Activation of nuclear receptor networks including the liver X receptor, farnesoid X receptor (FXR), peroxisome proliferator activated receptor, and retinoid X receptor (RXR) proteins have been found to play a key role in the induction of many genes responsible for both the transport and metabolism of bile acids (Chiang, 2002). Recently, reductions in the mRNA levels of several nuclear receptors [pregnane X receptor (PXR), constitutive androstane receptor (CAR) and FXR] were reported in rodents treated with endotoxin (Beigneux et al., 2000, 2002; Kalitsky-Szirtes et al., 2004). Moreover, it has been suggested that IL-1β-mediated down-regulation of MRP2 may occur, in part, through suppression of RXR (Denson et al., 2000, 2002). Thus, it is possible that nuclear receptor networks may play a role in the down-regulation of hepatic bile acid/salt transporters. However, little is known as to the involvement of nuclear receptors in the regulation of hepatic genes during inflammation.
Interestingly, many genes that are suppressed by endotoxin or other inflammatory stimuli have been shown to be induced by activation of PXR. It is well established that activation of PXR by hormones, bile acids, or xenobiotics leads to the induction of several genes including CYP3A (Bertilsson et al., 1998), OATP2 (Staudinger et al., 2003), MRP2 (Kast et al., 2002), MRP3 (Teng et al., 2003), and MDR1 (Geick et al., 2001). Studies in our laboratory have demonstrated that MDR1 and several members of the MRP and OATP families are down-regulated during inflammation (Hartmann et al., 2001, 2002; Lee and Piquette-Miller, 2001; Sukhai et al., 2001). Moreover, expression of CYP3A and other drug metabolizing enzymes are reduced under inflammatory conditions (Renton, 2001). Although studies have suggested the contribution of several transcription factors such as CCAAT/enhancer-binding protein (Jover et al., 2002) and HNF-1 (Memon et al., 2001; Roe et al., 2001), the mechanisms(s) involved in the down-regulation of these genes have not been firmly established. Furthermore, the potential involvement of PXR remains to be elucidated.
Hence, the main objective of this study was to investigate the role of the nuclear receptor PXR in mediating changes in the expression of hepatic bile salt/acid transporters and CYP3A during inflammation and to examine the specific cytokines involved. Findings from the study are the first to demonstrate involvement of PXR in BSEP regulation. Moreover, administration of the proinflammatory cytokine IL-6 reduced the hepatic expression of mrp2, bsep, and cyp3a11 in PXR+/+ but not PXR-/- mice and attenuated the PCN-mediated induction of mrp2, bsep, and cyp3a11. Hence, these novel findings suggest that, in addition to gene induction, PXR may play a role in the inflammation-mediated down-regulation of hepatic transporters and drug metabolizing enzymes.
Materials and Methods
Chemicals. PCN, RU486, endotoxin (lipopolysaccharide from Escherichia coli serotype 055:B5), and cytokines (recombinant mouse cytokines IL-6, IL-1β, TNF-α) were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada).
Animals. The animal studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care. Wild-type (PXR+/+) 8-week-old male C57BL/6 mice (25–30 g) were purchased from Charles River Canada (Montreal, PQ, Canada). PXR-null (PXR-/-) mice were kindly provided by Dr. Christopher Sinal (Dalhousie University, Halifax, NS) with permission from Dr. Steven Kliewer (University of Texas Southwestern Medical Center, Dallas, TX). Animals were kept in a temperature-controlled facility with 12-h light/dark cycles and were fed a standard chow diet.
To investigate the genes regulated by PXR, mice were administered daily the PXR activators PCN (50 mg/kg i.p., 4 days), RU486 (50 mg/kg i.m., 3 days), or corn oil vehicle control and were sacrificed by cervical dislocation 24 h following the last injection. The liver was removed, snap-frozen in liquid nitrogen, and stored at -80°C until analysis. These doses were not associated with hepatotoxicity as determined by normal serum alanine aminotransferase levels.
For the inflammation studies, mice were injected with endotoxin (5 or 7.5 mg/kg i.p.), IL-6 (1000–10,000 U i.p.), IL-1β (1000 or 10,000 U i.p.), TNF-α (10,000 or 25,000 U i.p.), or saline vehicle control and were sacrificed 6 h later. These doses were chosen because they were previously shown by our laboratory to be effective at modulating hepatic transporter expression in mice without causing hepatotoxicity (Hartmann et al., 2001, 2002). For studies of the reversibility of PXR-mediated transporter induction, the mice were injected each day for 4 days with PCN or corn oil as described above. On the 5th day, the mice received doses of endotoxin, IL-6, or saline vehicle control and were sacrificed 6 h later.
Determination of mRNA Expression. Total RNA was isolated from mouse liver using the QuickPrep RNA extraction kit (Amersham Biosciences Inc., Piscataway, NJ) according to manufacturer's protocol. cDNA was synthesized from 0.5 μg of RNA using the First Strand cDNA synthesis kit (MBI Fermentas, Flamborough, ON, Canada). In the PCN and RU486 activation studies, mRNA levels of various genes were determined by semiquantitative PCR with linear conditions established from standard curves as previously described (Hartmann et al., 2002). For the inflammation and cytokine studies, mRNA levels were measured by real-time quantitative PCR using LightCycler technology (Roche Diagnostics, Mannheim, Germany) with LC FastStart DNA Master SYBR Green I. All significant results from the PCN and RU486 studies were also confirmed using real-time PCR. Primers were synthesized by the DNA Synthesis Centre, Hospital for Sick Children (Toronto, ON, Canada). Primer sequences are listed in Table 1. All mRNA levels were normalized to gapdh mRNA. Normalization to either gapdh mRNA or 18S rRNA was found to give comparable results.
Determination of Transporter Protein Expression. The hepatic crude membrane fraction was isolated as described previously (Hartmann et al., 2001) and measured by the Bradford (1976) method using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). For the immunoblotting, 10 μg of protein was loaded and run on a 10% acrylamide gel and transferred onto a Hybond ECL nitrocellulose membrane (Amersham Biosciences Inc.). Membranes containing transporter protein were then cut in half and the upper portion (mol. wt. >78 kDa), was incubated with H-17 MRP2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by bovine anti-goat IgG (Santa Cruz Biotechnology, Inc.). To control for variability in protein loading, the lower portion of the membrane (mol. wt. <78 kDa) was incubated with anti-β-actin clone AC15 antibody (Sigma-Aldrich Canada) followed by sheep anti-mouse IgG (Amersham Biosciences Inc.). Bands were detected using an ECL Western blotting analysis system (Amersham Biosciences Inc.), imaged on Bioflex MSI film (Clonex Corp., InterSciences Inc., Markham, ON, Canada), and quantified using Kodak Digital Science 1D Image Analysis software (Eastman Kodak, Rochester, NY).
Determination of PXR Protein Expression. Immunodetectable levels of PXR were examined in hepatic nuclear proteins isolated from endotoxin (5 mg/kg), IL-6 (10,000 U), or saline control mice 6 h after treatment. Livers were homogenized in ice-cold buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.3 mM sucrose, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM sodium fluoride, and 0.1% Nonidet P-40) and centrifuged at 3000 rpm for 20 min. Pellets were incubated on ice for 1.5 h in high salt buffer and centrifuged at 14,000 rpm for 20 min. The supernatant was dialyzed for 2 h against 20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 20% glycerol. The dialysate was centrifuged at 14,000 rpm for 20 min, and the supernatant was collected and stored at -70°C until analysis. Immunoblotting for PXR was performed as described above using the R-14 antibody (Santa Cruz Biotechnology, Inc.).
Statistical Analysis. All studies were performed using n = 4 or more. Differences between PXR+/+ and PXR-/- mice and between treatment groups were determined by analysis of variance, and significance was determined by the Tukey test with p < 0.05 considered to be statistically significant. Analysis was performed using SigmaStat 2.03 (SPSS Inc., Chicago, IL).
Examination of the Effect of Endotoxin and Cytokines on PXR. To determine whether PXR expression is affected during an acute phase response, the effects of endotoxin (lipopolysaccharide) and proinflammatory cytokines on PXR were examined in vivo in PXR+/+ mice. As shown in Fig. 1, administration of 5 mg/kg endotoxin caused a significant down-regulation of PXR mRNA levels to 48 ± 9% of controls at 6 h. Similar changes were seen after administration of 7.5 mg/kg. IL-6 also elicited a dose-dependent down-regulation of PXR to 48 ± 12% of controls using 10,000 U. Likewise, immunodetectable levels of PXR were decreased to 38 ± 14% and 54 ± 10% of controls by endotoxin and IL-6, respectively (Fig. 1B). On the other hand, PXR mRNA was not significantly altered after administration of increasing doses of either IL-1β or TNF-α.
Identification of Transporter Genes Induced by PXR Activation. To identify the transporters regulated by PXR, both PXR wild-type (PXR+/+) and knock-out (PXR-/-) mice were treated with the PXR activators PCN or RU486. Compared with PXR+/+, basal levels of mdr2 (1.5-fold higher), mrp2 (1.5-fold higher), mrp3 (3-fold higher), and cyp3a (2-fold higher) were significantly elevated in the PXR-/- mice. As shown in Fig. 2, PCN and RU486 treatments imposed a significant induction in the mRNA levels of the ABC transporters bsep, mdr1a, mrp2, and mrp3 in PXR+/+ but not PXR-/- mice. Likewise, PXR activation with PCN or RU486 was found to significantly induce mRNA levels of the baso-lateral transporter oatp2 in PXR+/+ but not PXR-/- mice. This demonstrates a role for PXR in the regulation of bsep, mdr1a, mrp2, mrp3, and oatp2. Western blots also confirmed induction of MRP2 protein levels in PCN-treated PXR+/+ but not PXR-/- mice (Fig. 3A). On the other hand, the mRNA levels of the transporters bcrp (breast cancer resistance protein), mdr1b, mdr2, and ntcp were not significantly affected by PCN or RU486 treatments in either PXR+/+ or PXR-/- mice. As controls to verify our animal and experimental models, we measured the expression of cyp3a11 and cyp7a1, which are known to be induced and suppressed by PXR activation, respectively. As expected, mRNA levels of cyp3a11 were significantly induced and cyp7a1 significantly decreased in the PCN- and RU486-treated PXR+/+ mice, whereas changes were not detected in the PXR-/- mice.
Comparison of Endotoxin-Mediated Effects in PXR+/+ and PXR-/- Mice. To determine the involvement of PXR in mechanism(s) of transporter down-regulation during inflammation, we compared the effect of endotoxin on the expression of several transporters in PXR+/+ versus PXR-/- mice. As shown in Table 2, the mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were all significantly down-regulated by endotoxin administration in PXR+/+ mice with declines ranging from 18 to 61% of controls. In PXR-/- mice treated with endotoxin, the down-regulation of mrp2 was significantly less (52 ± 7% of controls, p < 0.05) than in PXR+/+ mice (18 ± 4% of controls, p < 0.001). Moreover, MRP2 protein expression was suppressed in PXR+/+ but not in PXR-/- mice (Fig. 3b). On the other hand, mrp3, oatp2, bsep, ntcp, mdr2, and cyp3a11 mRNA were down-regulated to the same extent in PXR-/- mice as in PXR+/+ mice.
Comparison of Cytokine-Mediated Effects in PXR+/+ and PXR-/- Mice. Previous studies have identified IL-6 as a principle mediator in inflammation-mediated down-regulation of hepatic transporters (Hartmann et al., 2002). Because IL-6 was found to impose a significant and substantial suppression of PXR mRNA in mice, we next compared the impact of IL-6 on the expression of cyp3a11 and transporters in PXR+/+ versus PXR-/- mice. Administration of IL-6 to PXR+/+ mice imposed a 28 to 45% down-regulation of bsep, mrp2, and cyp3a11, whereas significant changes were not detected in IL-6-treated PXR-/- mice (Table 3). Furthermore, MRP2 protein levels were down-regulated in PXR+/+ but not in PXR-/- mice. Although the IL-6-mediated down-regulation of oatp2 was somewhat attenuated in PXR-/- mice, differences in oatp2 mRNA levels between PXR-/- mice and PXR+/+ mice did not reach statistical significance. IL-6 did not elicit a down-regulation of ntcp mRNA levels in either the PXR+/+ or PXR-/- mice.
Since IL-1β did not have a significant effect on PXR mRNA levels, we examined expression of the bsep, mrp2, and cyp3a11 genes in IL-1β-treated PXR+/+ and PXR-/- mice as a control for PXR-independent regulation. Indeed, although we detected down-regulation of these genes in the IL-1β-treated mice, the extent of down-regulation of bsep, mrp2, and cyp3a11 was similar in both PXR+/+ and PXR-/- mice (Table 4). In addition, as the down-regulation of ntcp by endotoxin occurs through IL-1β-mediated pathways and is dependent on transcription factors other than PXR, we also compared the effect of IL-1β on ntcp levels in PXR+/+ and PXR-/- mice to further confirm PXR-independent gene suppression by IL-1β. As expected, ntcp down-regulation was similar in both types of mice.
Because differences in down-regulation could be due to differences in the severity of the inflammation induced by endotoxin or IL-6, we examined the expression of two acute phase proteins in PXR+/+ and PXR-/- mice. The induction of serum amyloid A and metallothionein were measured and were found to be similar for both PXR+/+ and PXR-/- mice (3.5- to 5.8-fold induction, p < 0.001). Furthermore, because the substrate specificity and target genes of PXR overlap with those of FXR and CAR and each of these receptors requires heterodimer formation with RXR, compensatory increases in the expression of these other nuclear receptors in PXR-/- mice could contribute to differences in down-regulation between PXR+/+ and PXR-/- mice. However, this does not appear to occur as comparisons of basal expression of FXR, CAR, and RXR mRNA in PXR+/+ and PXR-/- mice did not detect any significant difference between these two groups of mice (data not shown).
Impact of Endotoxin or IL-6 in PCN-Treated Mice. To further investigate the relationship between inflammation and PXR-mediated regulation of transporter expression, we examined the effect of IL-6 administration on gene expression following PXR activation in PCN- and RU486-pretreated mice. As shown in Fig. 4, PXR mRNA was increased by PCN pretreatment, and this induction was attenuated by administration of either endotoxin or IL-6. Correspondingly, administration of IL-6 attenuated the induction of bsep, mrp2, and cyp3a11 but not oatp2 in PCN-pretreated mice. Although RU486 pretreatment imposed a smaller (1.4- to 1.6-fold) but significant induction of these genes, administration of IL-6 to these animals suppressed bsep, mrp2, and cyp3a11 expression by 60, 25, and 85%, respectively. Administration of endotoxin to PCN-pretreated mice also suppressed the induction of mrp2 and cyp3a11 mRNA.
Reductions in the transport of bile salts and acids have been shown to play a critical role in the development of endotoxin- or inflammation-induced cholestasis. Nuclear receptors play a key role in the regulation of genes responsible for the metabolism and transport of bile acids. Hence, the goal of this study was to investigate the role of the nuclear receptor PXR in mediating changes in the expression of hepatic bile salt/acid transporters during endotoxin- or cytokine-induced inflammation. In this study, we demonstrated that endotoxin imposed a down-regulation in the hepatic expression of PXR mRNA and protein in mice. As administration of IL-6, but not IL-1β or TNF-α, suppressed the mRNA and protein expression of PXR, these results suggest that IL-6 is primarily responsible for the endotoxin-mediated down-regulation of PXR. This is consistent with our previous studies and those of others which have observed lower PXR levels in vivo in endotoxin-treated rodents and in vitro in IL-6-treated hepatocytes (Pascussi et al., 2000; Beigneux et al., 2002; Fang et al., 2004; Kalitsky-Szirtes et al., 2004; Xu et al., 2004). Moreover, IL-6 has been shown to be a major contributor to the regulation of organic anion transporters during endotoxemia (Siewert et al., 2004). Because PXR is involved in the transcriptional activation of CYP3A and several hepatic transporters, it is possible that diminished basal levels of PXR protein available for binding to promoter regions could cause decreases in the transcription and expression of these genes. Indeed, in addition to the findings that PXR protein levels are suppressed at 6 h, we also found that the down-regulation of PXR precedes the suppression of transporters. Three hours after endotoxin treatment we observed significant reductions in PXR mRNA levels (36 ± 4% of controls), whereas mrp2 mRNA were slightly but not significantly diminished (data not shown). This further suggests that transporter levels are at least partially dependent on PXR expression.
To date, the involvement of PXR in gene down-regulation by endotoxin has not been directly demonstrated. As endotoxin-induced changes were examined in PXR+/+ versus PXR-/- mice, our study is the first to show that PXR does, indeed, play a part in gene down-regulation during inflammation. First, the reduction of mrp2 mRNA and protein expression after endotoxin administration was significantly less extensive in PXR-/- mice compared with PXR+/+ mice. Second, IL-6 significantly decreased mrp2 expression in PXR+/+ but not PXR-/- mice. Overall, these results indicate that mrp2 down-regulation during inflammation is, at least in part, PXR-dependent and that PXR-dependent and -independent factors are involved. That PXR may be one of numerous contributors is not surprising as mrp2 expression has been shown to be regulated by several transcription factors (Denson et al., 2002; Kast et al., 2002; Geier et al., 2003b). Indeed, IL-1β has been shown to play a role in mrp2 down-regulation in rats during cholestasis and could occur, in part, through nuclear receptors such as RXR (Denson et al., 2002).
The role of PXR in the regulation of two other important hepatic bile salt/acid transporters BSEP and NTCP was also examined. The BSEP is a canalicular efflux transporter of bile salts. Currently, little is known about the regulation of bsep other than it is highly dependent on FXR (Plass et al., 2002; Wagner et al., 2003); however, it has been hypothesized that suppression of bsep expression may contribute to bile acid accumulation during cholestasis (Zollner et al., 2001). Results from this study demonstrated that PXR activation induces bsep expression in PXR wild-type but not knock-out mice. What's more, we found that the suppression of bsep by IL-6 in PXR+/+ mice did not occur in PXR-/- mice and that PXR-mediated induction of bsep in PCN-treated mice could be attenuated by IL-6. Overall, our study is the first to demonstrate that PXR plays a role in the regulation of bsep by PXR activation and through down-regulation by IL-6. Hence, PXR likely plays a role in bile acid feedback regulation (Staudinger et al., 2001; Xie et al., 2001). On the other hand, our study also indicated that expression of the hepatic basolateral bile acid uptake transporter ntcp is neither affected by PXR activation in wild-type nor altered in PXR knock-out mice. Although regulation of ntcp via PXR has not previously been examined, cholestasis-induced changes have been frequently reported (Trauner et al., 1999). Our findings that endotoxin and IL-1β but not IL-6 decreases ntcp expression is in agreement with the findings of others (Geier et al., 2003a; Siewert et al., 2004) who showed that IL-1β but not IL-6 is an important mediator of ntcp suppression by endotoxin.
Our results also indicate that PXR plays a role in the down-regulation of the drug metabolizing enzyme cyp3a11. Although cyp3a11 was suppressed in both endotoxin-treated PXR+/+ and PXR-/- mice, administration of IL-6 resulted in the suppression of cyp3a11 mRNA in PXR+/+ but not PXR-/- mice. Likewise, IL-6 treatments attenuated the PXR-mediated induction of cyp3a11 in PCN-treated mice. This suggests involvement of PXR in IL-6 mediated suppression of cyp3a11. It has been reported that IL-6 but not TNF-α or IL-1β diminish rifampicin-mediated induction of CYP3A4 in vitro (Muntane-Relat et al., 1995). Conversely, during endotoxemia, IL-6 is thought to provide minimal contribution to cyp3a11 regulation (Siewert et al., 2000). It is likely that PXR-independent pathways predominate in endotoxemia. Indeed, it has been reported that cyp3a11 down-regulation during endotoxemia likely occurs via peroxisome proliferator-activated receptor α (Barclay et al., 1999). In addition, Xu et al. (2004) recently reported that reactive oxygen species produced by Kupffer cells are involved in CYP3A suppression by endotoxin. Thus, activity of other inflammatory mediators induced in endotoxemia likely mask IL-6-mediated effects. These results further emphasize the complexity of the signaling pathways involved in gene regulation following exposure to endotoxin.
In addition to requiring heterodimer formation with RXR, PXR has been shown to share much functional redundancy with the nuclear hormone receptors FXR and CAR. Hence, an enhanced expression of FXR, CAR, or RXR in PXR-/- mice could preserve levels of regulated genes during inflammation. Likewise, compensatory increases in several transporters could also occur in knockouts to preserve homeostasis. We found that although basal levels of FXR, CAR, and RXR were not significantly different between PXR-/- and PXR+/+ mice, basal levels of mrp2 and cyp3a11 were higher in PXR-/- than in PXR+/+ mice. This could possibly obscure relative changes in the expression of these genes. On the other hand, baseline levels of mrp3 were also higher in PXR-/- mice, yet the extent of down-regulation by endotoxin or IL-6 was not different from PXR+/+ mice. This indicates that higher initial gene expression does not necessarily correlate with a lesser impact of inflammation. Moreover, we observed a comparable induction of the acute phase proteins serum amyloid A and metallothionein in the PXR+/+ and PXR-/- mice after endotoxin or cytokine administration, indicating similarity in the effect and intensity of inflammation between the two strains.
Our findings indicated that PCN- and RU486-mediated activation of PXR induced mdr1a mRNA levels but did not significantly affect mdr1b expression. This suggests differential PXR-mediated regulation of mdr1a and mdr1b. This is consistent with the distinct regulation seen with other signaling pathways (Hartmann et al., 2001). Although MDR1 induction is commonly cited to occur via PXR activation, this has only been reported in vitro in human cell lines (Geick et al., 2001; Synold et al., 2001). A relatively new member of the ABC transporter family, the breast cancer resistance protein (ABCG2) is also thought to be involved in drug resistance (Miyake et al., 1999). Very little is known about the regulation of this canalicular transporter. Our results indicate that PXR does not play a role in BCRP expression because mRNA levels were not affected by PXR activation. Moreover, similar basal levels of expression were seen in PXR-/-. Clearly, further studies are necessary to characterize the regulation of this transporter.
IL-6 and endotoxin administration to wild-type mice were found to suppress hepatic expression of PXR as well as attenuate PXR-mediated gene induction. Moreover, as IL-6-mediated changes in hepatic gene expression were subdued in PXR knock-out mice, our data suggests that inflammatory-mediated changes in expression of PXR-regulated genes may be secondary to the loss of PXR. The mechanism of PXR suppression by inflammatory stimulants remains unknown. Results in our laboratory show that HNF-4α is suppressed to less than 50% of controls by IL-6 in both PXR+/+ and PXR-/- mice. Furthermore, we observed a significant correlation (r = 0.94, p < 0.001) between PXR and HNF-4α mRNA levels in control and IL-6-treated mice (data not shown). Others have shown that HNF-4α is an important determinant of PXR expression during development (Li et al., 2000; Kamiya et al., 2003) but can also be critical for PXR-mediated induction of CYP3A4 (Tirona et al., 2003). Thus, inflammation-induced changes in the expression of HNF-4α or its gene targets could be responsible for initiating changes in PXR expression, subsequently affecting the expression of PXR target genes. Studies of HNF-4α involvement are currently ongoing.
In summary, findings from this study demonstrate that PXR regulates the expression of numerous hepatic transporters. Moreover, since inflammation-mediated down-regulation of mrp2, bsep, and cyp3a11 are significantly altered in the absence of functional PXR, this nuclear receptor appears to play a role in the mechanism of down-regulation of these genes during inflammation. Although a decrease in PXR expression could also occur parallel to changes in hepatic genes, the fact that IL-6-mediated suppression of several of these genes did not occur in PXR-/- mice suggest that PXR is required for this signaling pathway. Hence, transporter down-regulation may result due to decreased binding of PXR to promoter regions and reduced transcription. Thus, PXR expression may also play a role in inflammation-mediated down-regulation of hepatic bile transporters. These results place further value on the potential of PXR as a target for developing treatments for cholestasis.
We acknowledge the technical assistance provided by Veronika Jekerle, Georgy Hartmann, Susan Park, and Jing-Hung Wang.
- Received August 12, 2004.
- Accepted September 28, 2004.
Funding for this study was provided by a grant from the Canadian Institutes of Health Research (CIHR). M.P.-M. and S.T. were both recipients of Canada's Research-Based Pharmaceutical Companies (Rx&D Health Research Foundation)-CIHR research awards.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NTCP, Na+ taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; BSEP, bile salt export pump; MRP, multidrug resistance associated protein; MDR, multidrug resistance; FXR, farnesoid X receptor; RXR, retinoid X receptor; PXR, pregnane X receptor; CAR, constitutive androstane receptor; IL, interleukin; HNF, hepatic nuclear factor; PCN, 5-pregnen-3β-ol-20-one-16α-carbonitrile; RU486, 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl] estra-4,9-dien-3-one; ABC, ATP-binding cassette; TNF, tumor necrosis factor; PCR, polymerase chain reaction; BCRP, breast cancer resistance protein; RT-PCR, reverse transcription-polymerase chain reaction.
- The American Society for Pharmacology and Experimental Therapeutics