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FIC1: another bile salt carrier within the enterohepatic circulation?

2001; Elsevier BV; Volume: 35; Issue: 4 Linguagem: Inglês

10.1016/s0168-8278(01)00182-9

1600-0641

Bruno Stieger,

Pharmacological Effects and Toxicity Studies

Undisturbed bile secretion is vital for normal liver function. Bile acids are the major solutes in bile. The primary bile acids, chenodeoxycholic acid (dihydroxylated) and cholic acid (trihydroxylated), are the predominant species and are synthesized from cholesterol. Their pKa values are about 6 and 5, respectively. After amidation at position C24 with glycine or taurine, the pKa value drops to approximately 4 for glycine conjugates and to below 2 for taurine conjugates [[1]Boyer J.L Meier P.J Characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles.Methods Enzymol. 1990; 192: 517-533Crossref PubMed Scopus (34) Google Scholar]. Due to their relatively high pKa values, unconjugated bile acids are protonated (i.e. uncharged) to a significant extent at physiological pH, and as such, may cross plasma membranes by diffusion. In contrast, because of their lower pKa values, conjugated bile acids are present predominantly in the anionic form, precluding them from diffusion across plasma membranes. Therefore, conjugated bile acids will be called bile salts. As a consequence of their detergent properties, bile salts promote aggregation of phospholipids and cholesterol within mixed micelles in bile and thereby provide a vehicle for the excretion of hydrophobic compounds via bile. Canalicular secretion of bile constituents involves the coordinated action of an array of different transport systems [[2]Jansen P.L.M Foreword: from classic bile physiology to cloned transporters.Semin Liver Dis. 2000; 20: 245-250PubMed Google Scholar]. Bile salts are then secreted via the bile duct into the duodenum where they promote absorption of dietary fats and lipid-soluble vitamins. Bile salts are almost quantitatively reabsorbed in the small intestine and transported back to the liver via portal blood for uptake into hepatocytes and resecretion into bile for enterohepatic circulation [[3]Bahar R.J Stolz A Bile acid transport.Gastroenterol Clin North Am. 1999; 28: 27-58Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. Canalicular bile salt secretion is mediated by the canalicular bile salt export pump (Bsep/BSEP, Abcb11 (rodents), ABCB11 (human)), which constitutes a key driving force in enterohepatic circulation. Impairment of canalicular secretion of major bile constituents (e.g. bile salts, phospholipids) leads to a reduction in bile flow or cholestasis [[4]Trauner M Meier P.J Boyer J.L Molecular pathogenesis of cholestasis.N Engl J Med. 1998; 339: 1217-1227Crossref PubMed Scopus (657) Google Scholar]. Cholestasis may be acquired, e.g. induced by sepsis, adverse drug reactions, pregnancy, or it may be inherited. Inherited forms of cholestasis or progressive familial intrahepatic cholestasis (PFIC) are subdivided into three forms [5Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Thompson R Jansen P.L.M Genetic defects in hepatocanalicular transport.Semin Liver Dis. 2000; 20: 365-372Crossref PubMed Scopus (44) Google Scholar]: PFIC1 (known as Byler's disease) patients have low γ-glutamyltransferase and high bile salt levels in serum and a secondary bile salt transport defect; PFIC2 (Byler's syndrome) patients also have low γ-glutamyltransferase and high bile salt levels in serum and a primary bile salt transport defect; PFIC3 patients have high γ-glutamyltransferase and moderate bile salt levels and a phospholipid transport defect. A gene locus for PFIC1 was mapped to 18q21–q22, while the locus for PFIC2 maps to chromosome 2q24 [[5]Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. The BSEP gene codes for the canalicular bile salt export pump BSEP [[7]Strautnieks S.S Bull L.N Knisely A.S Kocoshis S.A Dahl N Arnell H et al.A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.Nat Genet. 1998; 20: 233-238Crossref PubMed Scopus (841) Google Scholar]. The locus for PFIC3 maps to chromosome 7q21. Its gene product codes for MDR3 (ABCB4), a phosphatidylcholine translocator [[8]Oude Elferink R.P.J Groen A.K Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. While the biochemical function of the gene products causing PFIC2 and PCIC3 is well understood, the function of the gene causing PFIC1 has remained enigmatic so far. In contrast to PFIC1, which ultimately can lead to liver failure, benign recurrent intrahepatic cholestasis (BRIC) is an inherited form of cholestasis with recurrent episodes of intrahepatic cholestasis, which resolve spontaneously. High-resolution mapping of loci for PFIC1 and BRIC lead to the identification of a common mutated gene for both diseases: FIC1 [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar].In ref. [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar], Eppens et al. present a study on the expression of the gene product of FIC1. FIC1 was identified as a protein of 140 kDa molecular weight on Western blots from human small intestinal and liver samples with rabbit polyclonal antibodies raised against different peptides derived from FIC1. Morphologically, FIC1 was localized to the canalicular plasma membrane of hepatocytes in human and mouse liver. In liver samples from a patient with PFIC1, FIC1 could not be detected histochemically or on Western blots, while staining for p-glycoprotein using the C219 antibody was normal. Finally, FIC1 was also demonstrated at the apical plasma membrane of cholangiocytes on tissue sections from mouse liver. Thus, this study clearly localizes FIC1 to the canalicular plasma membrane of hepatocytes and provides strong evidence for its expression in the apical membrane domain of cholangiocytes. It clarifies the important issue of FIC1 expression in liver and clarifies previous, rather conflicting preliminary reports on the expression of FIC1 in rat hepatocytes [[11]Ujhazy P Ortiz D.F Misra J Li S Arias I.M ATP-dependent aminophospholipid translocase activity in rat canalicular membrane vesicles and its relationship to FIC1.Hepatology. 1999; 30: 462ACrossref PubMed Scopus (118) Google Scholar] and rat cholangiocytes [[12]Mutero A Bull L.N Pawlikowska L Freimer N.B Scharschmidt B.F Lomri N Cloning and tissue distribution of the rat homologue of the human gene involved in familial intrahepatic cholestasis type 1.Hepatology. 1998; 38: 530AGoogle Scholar]. From the present study, it may be inferred that FIC1 could also be localized in the apical membrane of enterocytes. It is very likely to be expressed in additional organs, since, on Northern blot, mRNA for FIC1 is identified in a wide variety of tissues [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar]. Patients with PFIC1 have elevated serum bile salts and very low amounts of bile salts in their bile [[5]Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. Hence, the canalicular expression of FIC1 is not unexpected if one assumes that it plays an important role in bile salt secretion and takes into account that mutations in its gene product lead to liver disease.The biochemical function of FIC1 has so far not been delineated. At this moment, the biochemical function of FIC1 can therefore not be explained beyond the level of speculation. Based on sequence comparison, FIC1 belongs to a subfamily of the protein class of P-type ATPases (ATP8B1) [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar]. This subfamily has been implicated in aminophospholipid translocase activity [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar]. Members of this family are ATPase II of bovine chromaffine granules [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar], the ATPase2 gene product of Plasmodium falciparum [[14]Trottein F Cowman A.F Molecular cloning and sequence of two novel P-type adenosinetriphosphatases from Plasmodium falciparum.Eur J Biochem. 1995; 227: 214-225Crossref PubMed Scopus (30) Google Scholar], the DRS2 gene product of yeast [[15]Ripmaster T.L Vaughn G.P Woolford J.L DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.Mol Cell Biol. 1993; 13: 7901-7912Crossref PubMed Scopus (96) Google Scholar], and the CELT24H7.5 locus of Caenorhabditis elegans [[16]Wilson R Ainscough R Anderson K Baynes C Berks M Bonfield J et al.2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar] and the gene product of human ATPase II [[17]Mouro I Halleck M.S Schlegel R.A Mattei M.G Williamson P Zachowski A et al.Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase.Biochem Biophys Res Commun. 1999; 257: 333-339Crossref PubMed Scopus (30) Google Scholar]. Based on experiments with yeast DRS2 null strains, it was postulated that the gene product of DRS2, and hence the other members of this family, would be an aminophospholipid translocase [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar]. However, a recent publication has cast doubt on the aminophospholipid translocase activity of the gene product of DRS2 [[18]Siegmund A Grant A Angeletti C Malone L Nichols J.W Rudolph H.K Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae.J Biol Chem. 1998; 273: 34399-34405Crossref PubMed Scopus (77) Google Scholar]. Two observations may provide a working hypothesis on the function of FIC1. First, besides its expression in different organs, FIC1 is highly expressed in the small intestine and to a lesser extent in the liver [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar], cholangiocytes [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar] and colon [[12]Mutero A Bull L.N Pawlikowska L Freimer N.B Scharschmidt B.F Lomri N Cloning and tissue distribution of the rat homologue of the human gene involved in familial intrahepatic cholestasis type 1.Hepatology. 1998; 38: 530AGoogle Scholar]. This could implicate a role of FIC1 in enterohepatic circulation of bile salts. Second, patients with PFIC1 secrete low amounts of bile salts and, interestingly, have a prominent reduction of the secretion of chenodeoxycholic relative to cholic acid conjugates in the residual bile salt pool secreted [19Tazawa Y Yamada M Nakagawa M Konno T Tada K Bile acid profiles in siblings with progressive intrahepatic cholestasis: absence of biliary chenodeoxycholate.J Pediatr Gastroenterol Nutr. 1985; 4: 32-37Crossref PubMed Scopus (38) Google Scholar, 20Jacquemin E Dumont M Bernard O Erlinger S Hadchouel M Evidence for defective primary bile acid secretion in children with progressive familial intrahepatic cholestasis (Byler disease).Eur J Pediatr. 1994; 153: 424-428Crossref PubMed Scopus (88) Google Scholar]. This seems to be compensated for by the secretion of hyocholate, which can be synthesized by 6α-hydroxylation of chenodeoxycholate, possibly by CYP3A4 [[21]Araya Z Wikvall K 6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes.Biochim Biophys Acta. 1999; 1438: 47-54Crossref PubMed Scopus (135) Google Scholar]. An earlier study already reported the appearance of unknown, trihydroxylated bile salts in bile and serum of PFIC1 patients [[22]Linarelli L.G Williams C.N Phillips M.J Byler's disease: fatal intrahepatic cholestasis.J Pediatr. 1972; 81: 484-492Abstract Full Text PDF PubMed Scopus (103) Google Scholar]. The marked reduction of hydrophobic bile salts in bile could indicate that FIC1 secretes highly hydrophobic bile salts, such as lithocholate [[23]Oude Elferink R.P.J van Berge Henegouwen G.P Cracking the genetic code for benign recurrent and progressive familial intrahepatic cholestasis.J Hepatol. 1998; 29: 317-320Abstract Full Text PDF PubMed Scopus (19) Google Scholar]. Administration of lithocholate and taurolithocholate to rats leads to cholestasis [[24]Kakis G Yousef I.M Pathogenesis of lithocholate- and taurolithocholate-induced intrahepatic cholestasis in rats.Gastroenterology. 1978; 75: 595-607Abstract Full Text PDF PubMed Google Scholar]. Therefore, accumulation of highly hydrophobic bile salts in hepatocytes could lead to cholestasis in patients with PFIC1. Even though the human bile salt export pump BSEP has not yet been functionally characterized, rat and mouse Bsep both transport taurochenodeoxycholate [25Stieger B Fattinger K Madon J Kullak Ublick G.A Meier P.J Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 26Noe J Hagenbuch B Meier P.J St-Pierre M.V Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Crossref PubMed Scopus (100) Google Scholar]. It is therefore highly likely that human BSEP also transports taurochenodeoxycholate. However, it is important to realize that there are species differences in affinities of bile salts to Bsep as seen by a comparison of data for rat [[25]Stieger B Fattinger K Madon J Kullak Ublick G.A Meier P.J Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar] and mouse Bsep [26Noe J Hagenbuch B Meier P.J St-Pierre M.V Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Crossref PubMed Scopus (100) Google Scholar, 27Green R.M Hoda F Ward K.L Molecular cloning and characterization of the murine bile salt export pump.Gene. 2000; 241: 117-123Crossref PubMed Scopus (112) Google Scholar]. In a system where multiple transport systems for a substrate are present, affinity plays a decisive role for determining the route taken. That BSEP transports conjugates of chenodeoxycholic acid becomes evident in patients with PFIC2, who have no functional BSEP and secrete virtually no bile salts [[28]Jansen P.L.M Strautnieks S.S Jacquemin E Hadchouel M Sokal E.M Hooiveld G.J et al.Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis.Gastroenterology. 1999; 117: 1370-1379Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Of note, in one of the patients presented in this study, lithocholate was found in its residual bile. This may present circumstantial evidence for an additional transport mechanism for hydrophobic bile salts, e.g. FIC1. Given the expression of FIC1 in canaliculi, the apical membrane of cholangiocytes, and putatively also on the apical membrane of the small intestine, its function seems to be to eliminate a hydrophobic substance from enterohepatic circulation by excreting it across the canalicular plasma membrane, to prevent re-entry of this hydrophobic compound via the apical membrane of cholangiocytes into the cholehepatic shunt pathway and to prevent re-entry into enterohepatic circulation via the cells lining the intestinal epithelium. Whether this unknown substance is indeed a hydrophobic bile salt (in conjugated or unconjugated form) remains to be investigated. Unconjugated bile acids tend to have a high diffusive component as a consequence of their relatively high pKa value, while conjugated bile salts certainly need a transporter. However, hydrophobic substances including bile salts may intercalate into the lipid bilayer with their uncharged moiety, and may hence require a transporter (or a flippase) for removal. The observation that bile diversion improves the condition of PFIC patients [29Emond J.C Whitington P.F Selective surgical management of progressive familial intrahepatic cholestasis (Byler's disease).J Pediatr Surg. 1995; 30: 1635-1641Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 30Rebhandl W Felberbauer F.X Turnbull J Paya K Barcik U Huber W.D et al.Biliary diversion by use of the appendix (cholecystoappendicostomy) in progressive familial intrahepatic cholestasis.J Pediatr Gastroenterol Nutr. 1999; 28: 217-219Crossref PubMed Scopus (22) Google Scholar] supports the idea that the substrate of FIC1 is not a common bile salt but rather another hydrophobic substance or a hydrophobic bile salt. The high expression level of FIC1 in the small intestine may protect enterocytes from damage inflicted by highly hydrophobic substances, such as constituents of diet or hydrophobic bile salts produced by intestinal bacteria. The high expression level of FIC1 in the intestine could explain the observation that diarrhea, as observed in PFIC1 patients [22Linarelli L.G Williams C.N Phillips M.J Byler's disease: fatal intrahepatic cholestasis.J Pediatr. 1972; 81: 484-492Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 31Clayton R.J Iber F.L Ruebner B.H McKusick V.A Byler disease. Fatal familial intrahepatic cholestasis in an Amish kindred.Am J Dis Child. 1969; 117: 112-124Crossref PubMed Scopus (259) Google Scholar, 32Bourke B Goggin N Walsh D Kennedy S Setchell K.D Drumm B Byler-like familial cholestasis in an extended kindred.Arch Dis Child. 1996; 75: 223-227Crossref PubMed Scopus (43) Google Scholar], does not resolve after liver transplantation [9Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar, 33Knisely A.S Progressive familial intrahepatic cholestasis: a personal perspective.Pediatr Dev Pathol. 2000; 3: 113-125Crossref PubMed Google Scholar].While the paper by Eppens et al. [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar] has now clarified important aspects regarding the expression of FIC1, knowledge about the biochemical function of FIC1 is still missing. Part of the difficulty in identifying its function may be the fact that defective FIC1 seems to have an indirect effect on important canalicular transporters such as BSEP. A more detailed comparative analysis of the bile constituents in bile from PFIC1 patients with normal bile might provide additional clues for the function of FIC1. Generation of FIC1 knockout mice should also shed light on the physiological function, even though the example of Bsep knockout mice demonstrates that these animal models not only answer questions but also raise new ones: while PFIC2 in humans is a severe liver disease, Bsep knockout mice continue to secrete species specific bile salt derivatives into bile and have non-progressive cholestasis [[34]Wang R Salem M Yousef I.M Tuchweber B Lam P Childs S.J et al.Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc Natl Acad Sci USA. 2001; 98: 2011-2016Crossref PubMed Scopus (280) Google Scholar]. Ultimately, heterologous expression of FIC1 is required to directly demonstrate its physiological (transport) function. Undisturbed bile secretion is vital for normal liver function. Bile acids are the major solutes in bile. The primary bile acids, chenodeoxycholic acid (dihydroxylated) and cholic acid (trihydroxylated), are the predominant species and are synthesized from cholesterol. Their pKa values are about 6 and 5, respectively. After amidation at position C24 with glycine or taurine, the pKa value drops to approximately 4 for glycine conjugates and to below 2 for taurine conjugates [[1]Boyer J.L Meier P.J Characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles.Methods Enzymol. 1990; 192: 517-533Crossref PubMed Scopus (34) Google Scholar]. Due to their relatively high pKa values, unconjugated bile acids are protonated (i.e. uncharged) to a significant extent at physiological pH, and as such, may cross plasma membranes by diffusion. In contrast, because of their lower pKa values, conjugated bile acids are present predominantly in the anionic form, precluding them from diffusion across plasma membranes. Therefore, conjugated bile acids will be called bile salts. As a consequence of their detergent properties, bile salts promote aggregation of phospholipids and cholesterol within mixed micelles in bile and thereby provide a vehicle for the excretion of hydrophobic compounds via bile. Canalicular secretion of bile constituents involves the coordinated action of an array of different transport systems [[2]Jansen P.L.M Foreword: from classic bile physiology to cloned transporters.Semin Liver Dis. 2000; 20: 245-250PubMed Google Scholar]. Bile salts are then secreted via the bile duct into the duodenum where they promote absorption of dietary fats and lipid-soluble vitamins. Bile salts are almost quantitatively reabsorbed in the small intestine and transported back to the liver via portal blood for uptake into hepatocytes and resecretion into bile for enterohepatic circulation [[3]Bahar R.J Stolz A Bile acid transport.Gastroenterol Clin North Am. 1999; 28: 27-58Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. Canalicular bile salt secretion is mediated by the canalicular bile salt export pump (Bsep/BSEP, Abcb11 (rodents), ABCB11 (human)), which constitutes a key driving force in enterohepatic circulation. Impairment of canalicular secretion of major bile constituents (e.g. bile salts, phospholipids) leads to a reduction in bile flow or cholestasis [[4]Trauner M Meier P.J Boyer J.L Molecular pathogenesis of cholestasis.N Engl J Med. 1998; 339: 1217-1227Crossref PubMed Scopus (657) Google Scholar]. Cholestasis may be acquired, e.g. induced by sepsis, adverse drug reactions, pregnancy, or it may be inherited. Inherited forms of cholestasis or progressive familial intrahepatic cholestasis (PFIC) are subdivided into three forms [5Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Thompson R Jansen P.L.M Genetic defects in hepatocanalicular transport.Semin Liver Dis. 2000; 20: 365-372Crossref PubMed Scopus (44) Google Scholar]: PFIC1 (known as Byler's disease) patients have low γ-glutamyltransferase and high bile salt levels in serum and a secondary bile salt transport defect; PFIC2 (Byler's syndrome) patients also have low γ-glutamyltransferase and high bile salt levels in serum and a primary bile salt transport defect; PFIC3 patients have high γ-glutamyltransferase and moderate bile salt levels and a phospholipid transport defect. A gene locus for PFIC1 was mapped to 18q21–q22, while the locus for PFIC2 maps to chromosome 2q24 [[5]Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. The BSEP gene codes for the canalicular bile salt export pump BSEP [[7]Strautnieks S.S Bull L.N Knisely A.S Kocoshis S.A Dahl N Arnell H et al.A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.Nat Genet. 1998; 20: 233-238Crossref PubMed Scopus (841) Google Scholar]. The locus for PFIC3 maps to chromosome 7q21. Its gene product codes for MDR3 (ABCB4), a phosphatidylcholine translocator [[8]Oude Elferink R.P.J Groen A.K Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. While the biochemical function of the gene products causing PFIC2 and PCIC3 is well understood, the function of the gene causing PFIC1 has remained enigmatic so far. In contrast to PFIC1, which ultimately can lead to liver failure, benign recurrent intrahepatic cholestasis (BRIC) is an inherited form of cholestasis with recurrent episodes of intrahepatic cholestasis, which resolve spontaneously. High-resolution mapping of loci for PFIC1 and BRIC lead to the identification of a common mutated gene for both diseases: FIC1 [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar]. In ref. [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar], Eppens et al. present a study on the expression of the gene product of FIC1. FIC1 was identified as a protein of 140 kDa molecular weight on Western blots from human small intestinal and liver samples with rabbit polyclonal antibodies raised against different peptides derived from FIC1. Morphologically, FIC1 was localized to the canalicular plasma membrane of hepatocytes in human and mouse liver. In liver samples from a patient with PFIC1, FIC1 could not be detected histochemically or on Western blots, while staining for p-glycoprotein using the C219 antibody was normal. Finally, FIC1 was also demonstrated at the apical plasma membrane of cholangiocytes on tissue sections from mouse liver. Thus, this study clearly localizes FIC1 to the canalicular plasma membrane of hepatocytes and provides strong evidence for its expression in the apical membrane domain of cholangiocytes. It clarifies the important issue of FIC1 expression in liver and clarifies previous, rather conflicting preliminary reports on the expression of FIC1 in rat hepatocytes [[11]Ujhazy P Ortiz D.F Misra J Li S Arias I.M ATP-dependent aminophospholipid translocase activity in rat canalicular membrane vesicles and its relationship to FIC1.Hepatology. 1999; 30: 462ACrossref PubMed Scopus (118) Google Scholar] and rat cholangiocytes [[12]Mutero A Bull L.N Pawlikowska L Freimer N.B Scharschmidt B.F Lomri N Cloning and tissue distribution of the rat homologue of the human gene involved in familial intrahepatic cholestasis type 1.Hepatology. 1998; 38: 530AGoogle Scholar]. From the present study, it may be inferred that FIC1 could also be localized in the apical membrane of enterocytes. It is very likely to be expressed in additional organs, since, on Northern blot, mRNA for FIC1 is identified in a wide variety of tissues [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar]. Patients with PFIC1 have elevated serum bile salts and very low amounts of bile salts in their bile [[5]Jacquemin E Hadchouel M Genetic basis of progressive familial intrahepatic cholestasis.J Hepatol. 1999; 31: 377-381Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar]. Hence, the canalicular expression of FIC1 is not unexpected if one assumes that it plays an important role in bile salt secretion and takes into account that mutations in its gene product lead to liver disease. The biochemical function of FIC1 has so far not been delineated. At this moment, the biochemical function of FIC1 can therefore not be explained beyond the level of speculation. Based on sequence comparison, FIC1 belongs to a subfamily of the protein class of P-type ATPases (ATP8B1) [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar]. This subfamily has been implicated in aminophospholipid translocase activity [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar]. Members of this family are ATPase II of bovine chromaffine granules [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar], the ATPase2 gene product of Plasmodium falciparum [[14]Trottein F Cowman A.F Molecular cloning and sequence of two novel P-type adenosinetriphosphatases from Plasmodium falciparum.Eur J Biochem. 1995; 227: 214-225Crossref PubMed Scopus (30) Google Scholar], the DRS2 gene product of yeast [[15]Ripmaster T.L Vaughn G.P Woolford J.L DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.Mol Cell Biol. 1993; 13: 7901-7912Crossref PubMed Scopus (96) Google Scholar], and the CELT24H7.5 locus of Caenorhabditis elegans [[16]Wilson R Ainscough R Anderson K Baynes C Berks M Bonfield J et al.2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar] and the gene product of human ATPase II [[17]Mouro I Halleck M.S Schlegel R.A Mattei M.G Williamson P Zachowski A et al.Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase.Biochem Biophys Res Commun. 1999; 257: 333-339Crossref PubMed Scopus (30) Google Scholar]. Based on experiments with yeast DRS2 null strains, it was postulated that the gene product of DRS2, and hence the other members of this family, would be an aminophospholipid translocase [[13]Tang X Halleck M.S Schlegel R.A Williamson P A subfamily of P-type ATPases with aminophospholipid transporting activity.Science. 1996; 272: 1495-1497Crossref PubMed Scopus (417) Google Scholar]. However, a recent publication has cast doubt on the aminophospholipid translocase activity of the gene product of DRS2 [[18]Siegmund A Grant A Angeletti C Malone L Nichols J.W Rudolph H.K Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae.J Biol Chem. 1998; 273: 34399-34405Crossref PubMed Scopus (77) Google Scholar]. Two observations may provide a working hypothesis on the function of FIC1. First, besides its expression in different organs, FIC1 is highly expressed in the small intestine and to a lesser extent in the liver [[9]Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar], cholangiocytes [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar] and colon [[12]Mutero A Bull L.N Pawlikowska L Freimer N.B Scharschmidt B.F Lomri N Cloning and tissue distribution of the rat homologue of the human gene involved in familial intrahepatic cholestasis type 1.Hepatology. 1998; 38: 530AGoogle Scholar]. This could implicate a role of FIC1 in enterohepatic circulation of bile salts. Second, patients with PFIC1 secrete low amounts of bile salts and, interestingly, have a prominent reduction of the secretion of chenodeoxycholic relative to cholic acid conjugates in the residual bile salt pool secreted [19Tazawa Y Yamada M Nakagawa M Konno T Tada K Bile acid profiles in siblings with progressive intrahepatic cholestasis: absence of biliary chenodeoxycholate.J Pediatr Gastroenterol Nutr. 1985; 4: 32-37Crossref PubMed Scopus (38) Google Scholar, 20Jacquemin E Dumont M Bernard O Erlinger S Hadchouel M Evidence for defective primary bile acid secretion in children with progressive familial intrahepatic cholestasis (Byler disease).Eur J Pediatr. 1994; 153: 424-428Crossref PubMed Scopus (88) Google Scholar]. This seems to be compensated for by the secretion of hyocholate, which can be synthesized by 6α-hydroxylation of chenodeoxycholate, possibly by CYP3A4 [[21]Araya Z Wikvall K 6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes.Biochim Biophys Acta. 1999; 1438: 47-54Crossref PubMed Scopus (135) Google Scholar]. An earlier study already reported the appearance of unknown, trihydroxylated bile salts in bile and serum of PFIC1 patients [[22]Linarelli L.G Williams C.N Phillips M.J Byler's disease: fatal intrahepatic cholestasis.J Pediatr. 1972; 81: 484-492Abstract Full Text PDF PubMed Scopus (103) Google Scholar]. The marked reduction of hydrophobic bile salts in bile could indicate that FIC1 secretes highly hydrophobic bile salts, such as lithocholate [[23]Oude Elferink R.P.J van Berge Henegouwen G.P Cracking the genetic code for benign recurrent and progressive familial intrahepatic cholestasis.J Hepatol. 1998; 29: 317-320Abstract Full Text PDF PubMed Scopus (19) Google Scholar]. Administration of lithocholate and taurolithocholate to rats leads to cholestasis [[24]Kakis G Yousef I.M Pathogenesis of lithocholate- and taurolithocholate-induced intrahepatic cholestasis in rats.Gastroenterology. 1978; 75: 595-607Abstract Full Text PDF PubMed Google Scholar]. Therefore, accumulation of highly hydrophobic bile salts in hepatocytes could lead to cholestasis in patients with PFIC1. Even though the human bile salt export pump BSEP has not yet been functionally characterized, rat and mouse Bsep both transport taurochenodeoxycholate [25Stieger B Fattinger K Madon J Kullak Ublick G.A Meier P.J Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 26Noe J Hagenbuch B Meier P.J St-Pierre M.V Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Crossref PubMed Scopus (100) Google Scholar]. It is therefore highly likely that human BSEP also transports taurochenodeoxycholate. However, it is important to realize that there are species differences in affinities of bile salts to Bsep as seen by a comparison of data for rat [[25]Stieger B Fattinger K Madon J Kullak Ublick G.A Meier P.J Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar] and mouse Bsep [26Noe J Hagenbuch B Meier P.J St-Pierre M.V Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.Hepatology. 2001; 33: 1223-1231Crossref PubMed Scopus (100) Google Scholar, 27Green R.M Hoda F Ward K.L Molecular cloning and characterization of the murine bile salt export pump.Gene. 2000; 241: 117-123Crossref PubMed Scopus (112) Google Scholar]. In a system where multiple transport systems for a substrate are present, affinity plays a decisive role for determining the route taken. That BSEP transports conjugates of chenodeoxycholic acid becomes evident in patients with PFIC2, who have no functional BSEP and secrete virtually no bile salts [[28]Jansen P.L.M Strautnieks S.S Jacquemin E Hadchouel M Sokal E.M Hooiveld G.J et al.Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis.Gastroenterology. 1999; 117: 1370-1379Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar]. Of note, in one of the patients presented in this study, lithocholate was found in its residual bile. This may present circumstantial evidence for an additional transport mechanism for hydrophobic bile salts, e.g. FIC1. Given the expression of FIC1 in canaliculi, the apical membrane of cholangiocytes, and putatively also on the apical membrane of the small intestine, its function seems to be to eliminate a hydrophobic substance from enterohepatic circulation by excreting it across the canalicular plasma membrane, to prevent re-entry of this hydrophobic compound via the apical membrane of cholangiocytes into the cholehepatic shunt pathway and to prevent re-entry into enterohepatic circulation via the cells lining the intestinal epithelium. Whether this unknown substance is indeed a hydrophobic bile salt (in conjugated or unconjugated form) remains to be investigated. Unconjugated bile acids tend to have a high diffusive component as a consequence of their relatively high pKa value, while conjugated bile salts certainly need a transporter. However, hydrophobic substances including bile salts may intercalate into the lipid bilayer with their uncharged moiety, and may hence require a transporter (or a flippase) for removal. The observation that bile diversion improves the condition of PFIC patients [29Emond J.C Whitington P.F Selective surgical management of progressive familial intrahepatic cholestasis (Byler's disease).J Pediatr Surg. 1995; 30: 1635-1641Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 30Rebhandl W Felberbauer F.X Turnbull J Paya K Barcik U Huber W.D et al.Biliary diversion by use of the appendix (cholecystoappendicostomy) in progressive familial intrahepatic cholestasis.J Pediatr Gastroenterol Nutr. 1999; 28: 217-219Crossref PubMed Scopus (22) Google Scholar] supports the idea that the substrate of FIC1 is not a common bile salt but rather another hydrophobic substance or a hydrophobic bile salt. The high expression level of FIC1 in the small intestine may protect enterocytes from damage inflicted by highly hydrophobic substances, such as constituents of diet or hydrophobic bile salts produced by intestinal bacteria. The high expression level of FIC1 in the intestine could explain the observation that diarrhea, as observed in PFIC1 patients [22Linarelli L.G Williams C.N Phillips M.J Byler's disease: fatal intrahepatic cholestasis.J Pediatr. 1972; 81: 484-492Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 31Clayton R.J Iber F.L Ruebner B.H McKusick V.A Byler disease. Fatal familial intrahepatic cholestasis in an Amish kindred.Am J Dis Child. 1969; 117: 112-124Crossref PubMed Scopus (259) Google Scholar, 32Bourke B Goggin N Walsh D Kennedy S Setchell K.D Drumm B Byler-like familial cholestasis in an extended kindred.Arch Dis Child. 1996; 75: 223-227Crossref PubMed Scopus (43) Google Scholar], does not resolve after liver transplantation [9Bull L.N van Eijk M.J Pawlikowska L DeYoung J.A Juijn J.A Liao M et al.A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis.Nat Genet. 1998; 18: 219-224Crossref PubMed Scopus (611) Google Scholar, 33Knisely A.S Progressive familial intrahepatic cholestasis: a personal perspective.Pediatr Dev Pathol. 2000; 3: 113-125Crossref PubMed Google Scholar]. While the paper by Eppens et al. [[10]Eppens E.F van Mil S.W.C de Vree J.M.L Mok K.S Juijn J.A Oude Elferink R.P.J et al.FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.J Hepatol. 2001; 35: 436-443Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar] has now clarified important aspects regarding the expression of FIC1, knowledge about the biochemical function of FIC1 is still missing. Part of the difficulty in identifying its function may be the fact that defective FIC1 seems to have an indirect effect on important canalicular transporters such as BSEP. A more detailed comparative analysis of the bile constituents in bile from PFIC1 patients with normal bile might provide additional clues for the function of FIC1. Generation of FIC1 knockout mice should also shed light on the physiological function, even though the example of Bsep knockout mice demonstrates that these animal models not only answer questions but also raise new ones: while PFIC2 in humans is a severe liver disease, Bsep knockout mice continue to secrete species specific bile salt derivatives into bile and have non-progressive cholestasis [[34]Wang R Salem M Yousef I.M Tuchweber B Lam P Childs S.J et al.Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis.Proc Natl Acad Sci USA. 2001; 98: 2011-2016Crossref PubMed Scopus (280) Google Scholar]. Ultimately, heterologous expression of FIC1 is required to directly demonstrate its physiological (transport) function.