![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada
Received for publication
August 12, 2004
Accepted
September 28, 2004.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-ol-20-one-16
-carbonitrile (PCN) or 17-hydroxy-11-[4-dimethylamino phenyl]-17-[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.
). 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-1-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 |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
, TNF-) 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-1 (1000 or 10,000 U i.p.), TNF- (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--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).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
or TNF-.
|
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-1 did not have a significant effect on PXR mRNA levels, we examined expression of the bsep, mrp2, and cyp3a11 genes in IL-1-treated PXR+/+ and PXR-/- mice as a control for PXR-independent regulation. Indeed, although we detected down-regulation of these genes in the IL-1-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-1-mediated pathways and is dependent on transcription factors other than PXR, we also compared the effect of IL-1 on ntcp levels in PXR+/+ and PXR-/- mice to further confirm PXR-independent gene suppression by IL-1. 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.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
or TNF-, 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-1 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-1 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-1 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- or IL-1 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 (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-4 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-4 mRNA levels in control and IL-6-treated mice (data not shown). Others have shown that HNF-4 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-4 or its gene targets could be responsible for initiating changes in PXR expression, subsequently affecting the expression of PXR target genes. Studies of HNF-4 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.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
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-3-ol-20-one-16-carbonitrile; RU486, 17-hydroxy-11-[4-dimethylamino phenyl]-17-[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.
Address correspondence to: Dr. Micheline Piquette-Miller, Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada, M5S 2S2. E-mail: m.piquette.miller{at}utoronto.ca
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Barclay TB, Peters JM, Sewer MB, Ferrari L, Gonzalez FJ, and Morgan ET (1999) Modulation of cytochrome P-450 gene expression in endotoxemic mice is tissue specific and peroxisome proliferator-activated receptor-alpha dependent. J Pharmacol Exp Ther 290: 1250-1257.
Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR (2000) The acute phase response is associated with retinoid X receptor repression in rodent liver. J Biol Chem 275: 16390-16399.
Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR (2002) Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response. Biochem Biophys Res Commun 293: 145-149.[CrossRef][Medline]
Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P, and Berkenstam A (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci USA 95: 12208-12213.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Chiang JY (2002) Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev 23: 443-463.
Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, and Karpen SJ (2000) Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J Biol Chem 275: 8835-8843.
Denson LA, Bohan A, Held MA, and Boyer JL (2002) Organ-specific alterations in RAR alpha:RXR alpha abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis. Gastroenterology 123: 599-607.[CrossRef][Medline]
Fang C, Yoon S, Tindberg N, Jarvelainen HA, Lindros KO, and Ingelman-Sundberg M (2004) Hepatic expression of multiple acute phase proteins and down-regulation of nuclear receptors after acute endotoxin exposure. Biochem Pharmacol 67: 1389-1397.[CrossRef][Medline]
Geick A, Eichelbaum M, and Burk O (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem 276: 14581-14587.
Geier A, Dietrich CG, Voigt S, Kim SK, Gerloff T, Kullak-Ublick GA, Lorenzen J, Matern S, and Gartung C (2003a) Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis. Hepatology 38: 345-354.[CrossRef][Medline]
Geier A, Mertens PR, Gerloff T, Dietrich CG, En-Nia A, Kullak-Ublick GA, Karpen SJ, Matern S, and Gartung C (2003b) Constitutive rat multidrug-resistance protein 2 gene transcription is down-regulated by Y-box protein 1. Biochem Biophys Res Commun 309: 612-618.[CrossRef][Medline]
Hartmann G, Cheung AK, and Piquette-Miller M (2002) Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J Pharmacol Exp Ther 303: 273-281.
Hartmann G, Kim H, and Piquette-Miller M (2001) Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice. Int J Immunopharmacol 1: 189-199.
Jover R, Bort R, Gomez-Lechon MJ, and Castell JV (2002) Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved. FASEB J 16: 1799-1801.
Kalitsky-Szirtes J, Shayeganpour A, Brocks DR, and Piquette-Miller M (2004) Suppression of drug-metabolizing enzymes and efflux transporters in the intestine of endotoxin-treated rats. Drug Metab Dispos 32: 20-27.
Kamiya A, Inoue Y, and Gonzalez FJ (2003) Role of the hepatocyte nuclear factor 4alpha in control of the pregnane X receptor during fetal liver development. Hepatology 37: 1375-1384.[CrossRef][Medline]
Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, and Edwards PA (2002) Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor and constitutive androstane receptor. J Biol Chem 277: 2908-2915.
Lee G and Piquette-Miller M (2001) Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells. Can J Physiol Pharmacol 79: 876-884.[CrossRef][Medline]
Li J, Ning G, and Duncan SA (2000) Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev 14: 464-474.
Memon RA, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR (2001) In vivo and in vitro regulation of sterol 27-hydroxylase in the liver during the acute phase response potential role of hepatocyte nuclear factor-1. J Biol Chem 276: 30118-30126.
Miyake K, Mickley LA, Litman T, Greenberger L, Zhan Z, Robey R, Cristensen B, Brangi M, Greenberger L, Dean M, et al. (1999) Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells. Demonstration of homology to ABC transport genes. Cancer Res 59: 8-13.
Muntane-Relat J, Ourlin J, Domergue J, and Maurel P (1995) Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2 and CYP3A3 in human hepatocytes in primary culture. Hepatology 22: 1143-1153.[CrossRef][Medline]
Pascussi JM, Gerbal-Chaloin S, Pichard-Garcia L, Daujat M, Fabre JM, Maurel P, and Vilarem MJ (2000) Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes. Biochem Biophys Res Commun 274: 707-713.[CrossRef][Medline]
Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PL, and Muller M (2002) Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 35: 589-596.[CrossRef][Medline]
Renton KW (2001) Alteration of drug biotransformation and elimination during infection and inflammation. Pharmacol Ther 92: 147-163.[CrossRef][Medline]
Roe AL, Poloyac SM, Howard G, Shedlofsky SI, and Blouin RA (2001) The effect of endotoxin on hepatocyte nuclear factor 1 nuclear protein binding: potential implications on CYP2E1 expression in the rat. J Pharm Pharmacol 53: 1365-1371.[CrossRef][Medline]
Siewert E, Bort R, Kluge R, Heinrich PC, Castell J, and Jover R (2000) Hepatic cytochrome P450 down-regulation during aseptic inflammation in the mouse is interleukin 6 dependent. Hepatology 32: 49-55.[CrossRef][Medline]
Siewert E, Dietrich CG, Lammert F, Heinrich PC, Matern S, Gartung C, and Geier A (2004) Interleukin-6 regulates hepatic transporters during acute-phase response. Biochem Biophys Res Commun 322: 232-238.[CrossRef][Medline]
Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, MacKenzie KI, LaTour A, Liu Y, Klaassen CD, Brown KK, Reinhard J, et al. (2001) The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity. Proc Natl Acad Sci USA 98: 3369-3374.
Staudinger JL, Madan A, Carol KM, and Parkinson A (2003) Regulation of drug transporter gene expression by nuclear receptors. Drug Metab Dispos 31: 523-527.
Sukhai M, Yong A, Pak A, and Piquette-Miller M (2001) Decreased expression of P-glycoprotein in interleukin-1beta and interleukin-6 treated rat hepatocytes. Inflamm Res 50: 362-370.[CrossRef][Medline]
Synold TW, Dussault I, and Forman BM (2001) The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat Med 7: 584-590.[CrossRef][Medline]
Teng S, Jekerle V, and Piquette-Miller M (2003) Induction of ABCC3 (MRP3) by pregnane X receptor activators. Drug Metab Dispos 31: 1296-1299.
Tirona RG, Lee W, Leake BF, Lan LB, Cline CB, Lamba V, Parvis F, Duncan SA, Inoue Y, Gonzalez FJ, et al. (2003) The orphan nuclear receptor HNF4alpha determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat Med 9: 220-224.[CrossRef][Medline]
Trauner M, Fickert P, and Stauber RE (1999) Inflammation-induced cholestasis. J Gastroenterol Hepatol 14: 946-959.[CrossRef][Medline]
Wagner M, Fickert P, Zollner G, Fuchsbichler A, Silbert D, Tsybrovskyy O, Zatloukal K, Guo GL, Schuetz JD, Gonzalez FJ, et al. (2003) Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterology 125: 825-838.[CrossRef][Medline]
Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, Ong ES, Waxman DJ, and Evans RM (2001) An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proc Natl Acad Sci USA 98: 3375-3380.
Xu DX, Wei W, Sun MF, Wu CY, Wang JP, Wei LZ, and Zhou CF (2004) Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced down-regulation of nuclear receptor pregnane X receptor and its target gene CYP3A in mouse liver. Free Radic Biol Med 37: 10-22.[CrossRef][Medline]
Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, Ferenci P, Stauber RE, Krejs GJ, Denk H, et al. (2001) Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology 33: 633-646.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  K. Lichti-Kaiser, C. Xu, and J. L. Staudinger Cyclic AMP-dependent Protein Kinase Signaling Modulates Pregnane x Receptor Activity in a Species-specific Manner J. Biol. Chem., March 13, 2009; 284(11): 6639 - 6649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Petrovic, J.-H. Wang, and M. Piquette-Miller Effect of Endotoxin on the Expression of Placental Drug Transporters and Glyburide Disposition in Pregnant Rats Drug Metab. Dispos., September 1, 2008; 36(9): 1944 - 1950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. T. Morgan, K. B. Goralski, M. Piquette-Miller, K. W. Renton, G. R. Robertson, M. R. Chaluvadi, K. A. Charles, S. J. Clarke, M. Kacevska, C. Liddle, et al. Regulation of Drug-Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer Drug Metab. Dispos., February 1, 2008; 36(2): 205 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Le Vee, P. Gripon, B. Stieger, and O. Fardel Down-Regulation of Organic Anion Transporter Expression in Human Hepatocytes Exposed to the Proinflammatory Cytokine Interleukin 1{beta} Drug Metab. Dispos., February 1, 2008; 36(2): 217 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tanaka, C. Chen, J. M. Maher, and C. D. Klaassen Ischemia-Reperfusion of Rat Livers Decreases Liver and Increases Kidney Multidrug Resistance Associated Protein 2 (Mrp2) Toxicol. Sci., January 1, 2008; 101(1): 171 - 178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wagner, G. Zollner, P. Fickert, J. Gumhold, D. Silbert, A. Fuchsbichler, J. S. Gujral, K. Zatloukal, H. Denk, H. Jaeschke, et al. Hepatobiliary Transporter Expression in Intercellular Adhesion Molecule 1 Knockout and Fas Receptor-Deficient Mice after Common Bile Duct Ligation Is Independent of the Degree of Inflammation and Oxidative Stress Drug Metab. Dispos., September 1, 2007; 35(9): 1694 - 1699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. K. Choi, J. W. Yang, S. H. Roh, C. Y. Han, and K. W. Kang Induction of multidrug resistance associated protein 2 in tamoxifen-resistant breast cancer cells Endocr. Relat. Cancer, June 1, 2007; 14(2): 293 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fradette, J. Batonga, S. Teng, M. Piquette-Miller, and P. du Souich Animal Models of Acute Moderate Hypoxia Are Associated with a Down-Regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and Up-Regulation of CYP3A6 and P-glycoprotein in Liver Drug Metab. Dispos., May 1, 2007; 35(5): 765 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Petrovic, S. Teng, and M. Piquette-Miller REGULATION OF DRUG TRANSPORTERS: DURING INFECTION AND INFLAMMATION Mol. Interv., April 1, 2007; 7(2): 99 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Jung, D. J. Mangelsdorf, and U. A. Meyer Pregnane X Receptor Is a Target of Farnesoid X Receptor J. Biol. Chem., July 14, 2006; 281(28): 19081 - 19091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Richardson and E. T. Morgan Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 703 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Hartmann, V. Vassileva, and M. Piquette-Miller IMPACT OF ENDOTOXIN-INDUCED CHANGES IN P-GLYCOPROTEIN EXPRESSION ON DISPOSITION OF DOXORUBICIN IN MICE Drug Metab. Dispos., June 1, 2005; 33(6): 820 - 828. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
