ABC transporter regulation by bile acids: where PXR meets FXR

Abstract

The excretion of bile salts from the hepatocyte into the bile canaliculus is a critical step in bile formation. Bile salts conjugated with taurine or glycine are effluxed into bile via the bile salt export pump (Bsep, Abcb11), a member of the multidrug resistance (MDR) gene family [[1]Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Hepatic transport of bile salts.Semin Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (234) Google Scholar]. In cholestatic liver injury, the transport polarity of the hepatocyte is altered profoundly. Whereas Bsep expression is preserved following bile duct ligation in rats [[2]Lee J.M. Trauner M. Soroka C.J. Stieger B. Meier P.J. Boyer J.L. Expression of the bile salt export pump is maintained after chronic cholestasis in the rat.Gastroenterology. 2000; 118: 163-172Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar], obstructive cholestasis leads to decreased hepatic expression of the multidrug resistance associated protein 2 (Mrp2, Abcc2), the major canalicular efflux pump for anionic conjugates including bilirubin glucuronides [[3]Trauner M. Boyer J.L. Bile salt transporters: molecular characterization, function and regulation.Physiol Rev. 2003; 83: 633-671PubMed Google Scholar]. To compensate for decreased Mrp2 expression and function, the basolateral efflux pump Mrp3 is upregulated [4Soroka C.J. Lee J.M. Azzaroli F. Boyer J.L. Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.Hepatology. 2001; 33: 783-791Crossref PubMed Scopus (252) Google Scholar, 5Donner M.G. Keppler D. Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver.Hepatology. 2001; 34: 351-359Crossref PubMed Scopus (261) Google Scholar]. The decrease in hepatic Mrp2 expression is associated with decreased nuclear binding of the nuclear receptor dimer RARα:RXRα, a transcriptional activator of the Mrp2 gene [6Denson L.A. Auld K.L. Schiek D.S. McClure M.H. Mangelsdorf D.J. Karpen S.J. Interleukin-1β suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation.J Biol Chem. 2000; 275: 8835-8843Crossref PubMed Scopus (155) Google Scholar, 7Denson L.A. Bohan A. Held M.A. Boyer J.L. Organ-specific alterations in RARα:RXRα abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis.Gastroenterology. 2002; 123: 599-607Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. In contrast to the effects on hepatic Mrp2 expression, renal Mrp2 expression is preserved or even upregulated following bile duct ligation [7Denson L.A. Bohan A. Held M.A. Boyer J.L. Organ-specific alterations in RARα:RXRα abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis.Gastroenterology. 2002; 123: 599-607Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Lee J. Azzaroli F. Wang L. Soroka C.J. Gigliozzi A. Setchell K.D. et al.Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.Gastroenterology. 2001; 121: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar]. Mrp2 is expressed on the apical surface of renal proximal tubular cells and induction of renal Mrp2 during obstructive cholestasis could permit the urinary excretion of potentially toxic compounds that are normally excreted into bile. The mechanisms by which the three ABC transporters Bsep, Mrp2 and Mrp3 are regulated in cholestasis are only partly understood. Important evidence derives from bile acid feeding models in mice. CA (cholic acid) or UDCA (ursodeoxycholic acid) feeding was shown to induce hepatic expression of both Bsep and Mrp2 [[9]Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Pojer C. Zenz R. et al.Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver.Gastroenterology. 2001; 121: 170-183Abstract Full Text PDF PubMed Scopus (222) Google Scholar]. A major transcription factor involved in the regulation of gene expression by bile acids is the bile acid receptor/farnesoid X receptor (BAR/FXR), also termed NR1H4, RIP14 and HRP1 [[10]Chiang J.Y.L. Bile acid regulation of gene expression: roles of nuclear hormone receptors.Endocr Rev. 2002; 23: 443-463Crossref PubMed Scopus (376) Google Scholar]. FXR belongs to the NR1H family of orphan nuclear receptors and its natural ligands are bile acids, notably chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and lithocholic acid (LCA) [11Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. et al.Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2081) Google Scholar, 12Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar]. CA and UDCA appear to be only weak FXR ligands [11Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. et al.Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2081) Google Scholar, 12Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.Mol Cell. 1999; 3: 543-553Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar]. In view of the rapid increase in hepatic and systemic bile salt concentrations that characterizes early stages of cholestatic liver injury, a role of FXR in mediating the changes in gene expression that occur in cholestasis seems likely. Transporter genes that are transcriptionally induced by FXR include rodent and human Bsep/BSEP [13Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar, 14Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. Human bile salt export pump (BSEP) promoter is transactivated by the farnesoid X receptor/bile acid receptor (FXR/BAR).J Biol Chem. 2001; 276: 28857-28865Crossref PubMed Scopus (639) Google Scholar, 15Schuetz E.G. Strom S. Yasuda K. Lecureur V. Assem M. Brimer C. et al.Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450.J Biol Chem. 2001; 276: 39411-39418Crossref PubMed Scopus (343) Google Scholar, 16Gerloff T. Geier A. Roots I. Meier P.J. Gartung C. Functional analysis of the rat bile salt export pump gene promoter.Eur J Biochem. 2002; 269: 3495-3503Crossref PubMed Scopus (43) Google Scholar], rat Mrp2 [[17]Kast H.R. Goodwin B. Tarr P.T. Jones S.A. Anisfeld A.M. Stoltz C.M. et al.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. 2002; 277: 2908-2915Crossref PubMed Scopus (756) Google Scholar], the human organic anion transporting polypeptide OATP8 (SLC21A8) [[18]Jung D. Podvinec M. Meyer U.A. Mangelsdorf D.J. Fried M. Meier P.J. et al.Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor.Gastroenterology. 2002; 122: 1954-1966Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar] and the ileal bile acid-binding protein [[19]Grober J. Zaghini I. Fujii H. Jones S.A. Kliewer S.A. Willson T.M. et al.Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer.J Biol Chem. 1999; 274: 29749-29754Crossref PubMed Scopus (286) Google Scholar]. Transporter genes that are transcriptionally repressed by FXR include rat Ntcp (Slc10a1) [[20]Denson L.A. Sturm E. Echevarria W. Zimmerman T.L. Makishima M. Mangelsdorf D.J. et al.The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp.Gastroenterology. 2001; 121: 140-147Abstract Full Text PDF PubMed Scopus (359) Google Scholar], human OATP-C (SLC21A6) [[21]Jung D. Kullak-Ublick G.A. Hepatocyte nuclear factor 1 alpha: a key mediator of the effect of bile acids on gene expression.Hepatology. 2003; 37: 622-631Crossref PubMed Scopus (155) Google Scholar] and the mouse ileal bile acid transporter Asbt (Slc10a2) [[22]Chen F. Ma L. Dawson P.A. Sinal C.J. Sehayek E. Gonzalez F.J. et al.Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter.J Biol Chem. 2003; 278: 19909-19916Crossref PubMed Scopus (193) Google Scholar]. To investigate the exact contribution of FXR to bile acid-mediated changes in the expression of the ABC transporters Bsep, Mrp2 and Mrp3, the study by Zollner, Fickert and colleagues in this issue of the Journal compares the effect of bile acid feeding on transporter expression in wild-type mice and mice with a targeted disruption of FXR [[23]Zollner G. Fickert P. Fuchsbichler A. Silbert D. Wagner M. Arbeiter S. et al.Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine.J Hepatol. 2003; 39: 480-488Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar]. In agreement with previous studies [[13]Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar], expression of Bsep is induced by both CA and UDCA in FXR+/+ but not FXR−/− mice. Moreover, baseline expression of Bsep is much lower in FXR−/− mice, indicating that FXR is essential for the transcription of the Bsep gene. In contrast to Bsep, hepatic expression of Mrp2 is induced by CA and UDCA not only in FXR+/+ mice, but also in FXR−/− mice. This suggests that the effect of bile acids on Mrp2 gene expression in hepatocytes is largely FXR-independent, although Mrp2 induction by UDCA is less pronounced in FXR−/− mice. Bile acid feeding induces Mrp2 not only in hepatocytes, but also in the kidney and in the intestine, notably in duodenum. In fact renal Mrp2 is induced more strongly in FXR−/− mice than in FXR+/+ mice, possibly because renal bile salt throughput is much higher in FXR−/− mice [[13]Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1367) Google Scholar]. At the basolateral pole of the hepatocyte, Mrp3 is also induced by CA and UDCA feeding, with a more pronounced effect in FXR−/− than in wild-type mice. The authors also describe Mrp3 expression in the intestine and show a 3 fold increase in baseline Mrp3 expression in FXR−/− mice that cannot be further induced by CA or UDCA. In FXR+/+ mice, CA and UDCA feeding increases Mrp3 expression in the duodenum and colon. Finally the authors describe the effect of CA and UDCA feeding on liver histology and show that CA feeding to FXR−/− mice leads to enlarged hepatocytes and focal necrosis, whereas UDCA feeding to FXR−/− mice only produces mild steatosis. This underlines the differential effect of these two bile acids on hepatocyte integrity and confirms the non-toxic properties of UDCA even in mice that have markedly reduced Bsep function. What is happening in FXR−/− mice that could account for the effects observed by Zollner, Fickert and colleagues? A major consequence of disrupting the FXR gene is the decreased expression of Bsep, which predisposes the hepatocyte to cholestatic injury following a bile acid challenge with CA. Decreased Bsep expression and function leads to elevated intracellular and systemic bile acid concentrations, a condition that is aggravated further by exogenous administration of bile acids. The authors propose that the increases in Mrp2 and Mrp3 expression that ensue from bile acid feeding in FXR−/− mice could be accounted for by bile acid-mediated activation of another nuclear receptor, the pregnane X receptor (PXR, NR1I2). PXR is a promiscuous xenobiotic receptor that is activated by numerous structurally unrelated steroids, xenobiotics and drugs, as well as bile acids including LCA and UDCA [15Schuetz E.G. Strom S. Yasuda K. Lecureur V. Assem M. Brimer C. et al.Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450.J Biol Chem. 2001; 276: 39411-39418Crossref PubMed Scopus (343) Google Scholar, 24Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. et al.The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.Proc Natl Acad Sci USA. 2001; 98: 3369-3374Crossref PubMed Scopus (1107) Google Scholar, 25Xie W. Radominska Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. et al.An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.Proc Natl Acad Sci USA. 2001; 98: 3375-3380Crossref PubMed Scopus (668) Google Scholar]. PXR is highly expressed in the liver and at more moderate levels in the intestine [[26]Francis G.A. Fayard E. Picard F. Auwerx J. Nuclear receptors and the control of metabolism.Annu Rev Physiol. 2003; 65: 261-311Crossref PubMed Scopus (496) Google Scholar]. PXR is closely related to the constitutive androstane receptor (CAR) and shares common ligands and function [[27]Willson T.M. Kliewer S.A. PXR, CAR and drug metabolism.Nat Rev Drug Discov. 2002; 1: 259-266Crossref PubMed Scopus (396) Google Scholar]. The effects of CA or UDCA feeding on hepatic Mrp3 and renal Mrp2 expression in both FXR+/+ and FXR−/− mice are qualitatively the same as those that result from bile duct ligation [4Soroka C.J. Lee J.M. Azzaroli F. Boyer J.L. Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.Hepatology. 2001; 33: 783-791Crossref PubMed Scopus (252) Google Scholar, 5Donner M.G. Keppler D. Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver.Hepatology. 2001; 34: 351-359Crossref PubMed Scopus (261) Google Scholar, 8Lee J. Azzaroli F. Wang L. Soroka C.J. Gigliozzi A. Setchell K.D. et al.Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.Gastroenterology. 2001; 121: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar], indicating that PXR may indeed regulate hepatic Mrp3 and renal Mrp2 expression in cholestasis. In the case of hepatic Mrp2, the effects of bile acid feeding differ from those seen during obstructive cholestasis, the latter being associated with a strong decrease in Mrp2 expression [[3]Trauner M. Boyer J.L. Bile salt transporters: molecular characterization, function and regulation.Physiol Rev. 2003; 83: 633-671PubMed Google Scholar]. Decreased Mrp2 in chronic bile duct ligation results in part from decreased expression of RARα [[7]Denson L.A. Bohan A. Held M.A. Boyer J.L. Organ-specific alterations in RARα:RXRα abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis.Gastroenterology. 2002; 123: 599-607Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar] an effect that appears to override Mrp2 activation by PXR and FXR. It is likely that CA or UDCA feeding does not decrease RARα, explaining why the net effect of bile acid feeding on Mrp2 is one of induction. Regulation of Mrp3 is also complex and involves both PXR and CAR [28Cherrington N.J. Hartley D.P. Li N. Johnson D.R. Klaassen C.D. Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats.J Pharmacol Exp Ther. 2002; 300: 97-104Crossref PubMed Scopus (195) Google Scholar, 29Staudinger J.L. Madan A. Carol K.M. Parkinson A. Regulation of drug transporter gene expression by nuclear receptors.Drug Metab Dispos. 2003; 31: 523-527Crossref PubMed Scopus (130) Google Scholar]. Although hepatic Mrp3 expression is induced by selective activators of PXR and CAR, both the baseline expression of Mrp3, as well as inducibility of Mrp3 by the CAR ligand phenobarbital, is higher in PXR−/− mice [[29]Staudinger J.L. Madan A. Carol K.M. Parkinson A. Regulation of drug transporter gene expression by nuclear receptors.Drug Metab Dispos. 2003; 31: 523-527Crossref PubMed Scopus (130) Google Scholar]. Thus non-liganded PXR appears to play a net negative role in coregulating the shared PXR/CAR target gene Mrp3. The human MRP3 gene is induced by bile salts through direct binding of the bile acid-inducible α1-fetoprotein transcription factor (FTF) to the MRP3 promoter [[30]Inokuchi A. Hinoshita E. Iwamoto Y. Kohno K. Kuwano M. Uchiumi T. Enhanced expression of the human multidrug resistance protein 3 by bile salt in human enterocytes. A transcriptional control of a plausible bile acid transporter.J Biol Chem. 2001; 276: 46822-46829Crossref PubMed Scopus (99) Google Scholar]. The induction of intestinal Mrp3/MRP3 by bile acids contrasts with the observation that bile duct ligation in rats actually decreases the intestinal absorption rate of taurocholate [[31]Sauer P. Stiehl A. Fitscher B.A. Riedel H.D. Benz C. Kloters Plachky P. et al.Downregulation of ileal bile acid absorption in bile-duct-ligated rats.J Hepatol. 2000; 33: 2-8Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. The observations of Zollner, Fickert and colleagues are complemented by a recent study analyzing the effect of LCA feeding to FXR+/+ and FXR−/− mice. In contrast to CA feeding, which increased alanine aminotransferase (ALT) levels more strongly in FXR−/− than in FXR+/+ mice (1625 vs. 43 U/l, respectively) [[23]Zollner G. Fickert P. Fuchsbichler A. Silbert D. Wagner M. Arbeiter S. et al.Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine.J Hepatol. 2003; 39: 480-488Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar], LCA feeding led to higher aspartate aminotransferase (AST) levels in FXR+/+ than in FXR−/− mice (955 vs. 143 U/l) [[32]Kitada H. Miyata M. Nakamura T. Tozawa A. Honma W. Shimada M. et al.Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity.J Biol Chem. 2003; 278: 17838-17844Crossref PubMed Scopus (129) Google Scholar]. This discrepancy between CA and LCA feeding was also evident with respect to changes in transporter expression: no obvious changes in Mrp2, Mrp3 and Mrp4 mRNA levels were induced by LCA feeding in either FXR+/+ or FXR−/− mice [[32]Kitada H. Miyata M. Nakamura T. Tozawa A. Honma W. Shimada M. et al.Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity.J Biol Chem. 2003; 278: 17838-17844Crossref PubMed Scopus (129) Google Scholar]. The most likely explanation for the differential effects of CA and LCA is the marked increase in the hepatic sulfating activity for LCA and hydroxysteroid sulfotransferase 2a expression in the livers of FXR−/− mice. A 7.4-fold higher 3α-sulfated bile acid concentration was observed in the bile of FXR−/− mice, indicating that the liver can rapidly conjugate and excrete LCA sulfate, thereby lowering LCA levels in the livers of FXR−/− mice. It should be noted that whereas in the LCA study female mice were used, the CA feeding study described in this issue of the Journal [[23]Zollner G. Fickert P. Fuchsbichler A. Silbert D. Wagner M. Arbeiter S. et al.Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine.J Hepatol. 2003; 39: 480-488Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar] employed male mice that lack bile acid sulfating activity despite being resistant to LCA-induced toxicity [[32]Kitada H. Miyata M. Nakamura T. Tozawa A. Honma W. Shimada M. et al.Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity.J Biol Chem. 2003; 278: 17838-17844Crossref PubMed Scopus (129) Google Scholar]. What do we learn from the studies by Zollner, Fickert and colleagues for our understanding of cholestatic liver disease? Bile acid feeding in wild-type mice leads to a combined FXR/PXR pattern of gene induction, whereas in FXR−/− mice only the PXR effect is evident. Induction of PXR is a detoxifying mechanism that allows rapid excretion of toxic bile salts from the enterohepatic circulation despite deficient bile salt excretion into bile. Is the therapeutic effect of UDCA in cholestatic liver disease attributable to its role as a non-toxic PXR ligand? If this is the case, could selective activation of PXR by synthetic agonists be a future treatment of cholestatic liver disease? Or is UDCA the magic bullet because of the multiplicity of its biologic effects on hepatocyte function [[33]Paumgartner G. Beuers U. Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited.Hepatology. 2002; 36: 525-531Crossref PubMed Scopus (525) Google Scholar]? We will obtain answers when the effects of bile acid feeding to PXR−/− and PXR−/−/FXR−/− mice are investigated. This work was supported by grant 632-062773 from the Swiss National Science Foundation, Bern, Switzerland.