- Email: [email protected]
Inherited disorders of transport in the liver
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Inherited disorders of transport in the liver Richard Thompson* and Sandra Strautnieks There has been a recent explosion in our understanding of hepatic transport processes. Much of this has resulted from the investigation of human diseases involving the liver and the use of animal models. The physiological roles of many of these transporters have been well characterised previously but have, until now, been resistant to molecular cloning. Addresses Department of Child Health, Guy’s, King’s and St Thomas’ School of Medicine, King’s College Hospital, London, SE5 9RS, UK *e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:310–313 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette BSEP bile salt export pump cMOAT canalicular multi-specific organic anion transporter HDL high-density lipoprotein HHH hyperornithinemia–hyperammonemia–homocitrullinuria MDR multidrug resistance MRP MDR-associated protein PFIC progressive familial intrahepatic cholestasis PHHI persistent hyperinsulinemia hypoglycemia of infancy
Introduction The adult human liver weighs ~1.5 kg and has a blood flow of ~1.5 litres per minute. 80% of the blood flow occurs via the hepatic portal vein. This key aspect of hepatic anatomy is critical to many of its functions. The gastrointestinal tract is our largest source of exposure to exogenous compounds. Some of these substances are potentially harmful though many are, of course, nutrients. In the form in which they are absorbed from the gastrointestinal tract, however, even many nutrients are often not beneficial to most cells in the body. Furthermore, most cells in the body require a steady supply of substrates rather than surges following meals. The function of the liver in this respect is to remove from the blood a large proportion of what is absorbed from the gut prior to the blood circulating to the rest of the body. In order to achieve this function, a multitude of transporter polypeptides are expressed in the hepatocyte basolateral membrane. The liver also has an excretory function: bile is formed by a separate set of transporter molecules in the hepatocyte canalicular membrane. The main purpose of bile production would appear to be the need to excrete bile acids into the gastrointestinal tract to the liver to absorb fats efficiently. Most other biliary constituents are either protective or could be excreted, though with less efficiency, via another route such as the kidney. In addition to uptake and efflux mechanisms, an increasing number of intracellular transporters have been identified and their genes cloned — many of which are not hepatocyte-specific.
Many of the molecules underlying a number of key hepatic transport processes have eluded cloning and characterisation attempts until recently. The study of patients with apparent defects of hepatic transport, however, has recently advanced our knowledge greatly. This has been aided further by the quantity and quality of information being generated by the human genome project. Some of the important transport processes within the liver that have been elucidated in this way are reviewed here.
The ABC of hepatocyte canalicular transporters Mutations in four human canalicular transporters have been shown to underlie different human diseases. We discuss these later. However there seems to be a much greater redundancy in the basolateral transporters. Not only do most basolateral transporters have a wide substrate specificity but there also is a good deal of overlap. The consequence is that no human disease has yet been associated with a single hepatocyte-specific basolateral transporter. Four key canalicular transporters are members of the ATPbinding cassette (ABC) superfamily. Three are even more closely related and are members of the ABCB family, which until recently was known as the MDR (multidrug resistance) family. The original member of the family was MDR1, also known as P-glycoprotein [1]. The name for the gene encoding this protein has recently changed from PGY1 to ABCB1. The gene is located on chromosome 7q21.1 somewhat proximal to the best-known ABC transporter encoding gene — CFTR. As the name suggests, this protein was identified as a result of its ability to confer resistance to chemotherapy agents in tumour cells. In fact this function is not an epiphenomenon but probably derives from its true physiological function. It is capable of transporting a wide range of drugs. No human disease has so far been attributed to mutations in this gene, and the ‘knockout’ mouse is essentially normal [2]. However, drug handling in the murine model was far from normal both with respect to renal and hepatic excretion and also with respect to central nervous system penetration. MDR1 is certainly expressed in the blood–brain barrier. Polymorphic variations in ABCB1 probably confer variation in drug distribution and elimination in man. ABCB4 encodes MDR3 [3]. ABCB4 was known as PGY3 and the mouse gene product is confusingly known as mdr2. The human gene is also found on 7q21.1 close to ABCB1. Homozygous knockout mice for this gene were recognised to lack biliary phospholipid excretion [2]. It was subsequently realised that a group of patients with a severe form of liver disease had histological features similar to the knockout mice [4]. This disease has become known as ‘high γGT progressive familial intrahepatic cholestasis’ (high γGT PFIC). Both reduced levels of ABCB4 mRNA,
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and (subsequently) coding sequence mutations have been found in these patients [5]. As with the knockout mice they also either have dramatically reduced or absent biliary phospholipid excretion. Importantly, one of the mothers — an obligate heterozygote for an ABCB4 mutation — had cholestasis during pregnancy [5]. Subsequently, a large family has been reported in which four obligate heterozygote mothers and two other relatives had cholestasis during pregnancy [6]. The third member of the MDR family expressed in the canalicular membrane is the bile salt export pump (BSEP). When first identified the gene was termed ‘sister of P-glycoprotein’, it was then termed BSEP but has now been designated ABCB11. This gene was originally identified as a pig expressed sequence tag (EST), and subsequently cloned in rat [7] and human [8]. The later was undertaken after a form of low γGT PFIC was mapped by linkage analysis to the same locus [9]. These patients have very low levels of bile acids in their bile, despite normal bile acid synthesis and high levels in serum [10•]. The rat orthologue has been expressed in vitro and is a competent bile acid transporter with a Km of the order to be expected from previous physiology studies using canalicular membrane vesicles [7]. It has also been shown recently that there is a good correlation between mutations in ABCB11 and a lack of BSEP-specific immuno-staining of liver. Several patients also had biliary bile acid analysis performed, confirming the near total absence of excretion [10•]. The fourth ABC transporter present in the hepatocyte canalicular membrane is the canalicular multi-specific organic anion transporter (cMOAT). Again the gene name has recently changed, from CMOAT to ABCC2. The C family of ABC transporters are more heterogeneous in structure than the B family and include the cystic fibrosis transmembrane conductance regulator (encoded by ABCC7) and the sulfonylurea receptor (encoded by ABCC8). The latter is mutated in some cases of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). CMOAT is also known as multidrug-resistance-associated protein 2 (MRP2). It is not clear whether it is capable of conferring drug resistance but it does transport a wide range of conjugated anions across the canalicular membrane. The major endogenous substrates are bilirubin glucuronides. Autosomal recessive mutations have been identified in this gene in patients with Dubin–Johnson syndrome [11]. This mild disease is characterised by conjugated hyperbilirubinemia and the deposition of very dark pigment in the liver. The pigment is not bilirubin. In both Dubin–Johnson syndrome patients and the TRmutant and Eisai hyperbilrubinemic rat models [12], there is overexpression of another ABC transporter — MRP3 [13,14], which is encoded by ABCC3. It has been suggested that this overexpression is a compensatory mechanism allowing bilirubin efflux, though clearly not of the pigment that accumulates. There is conflicting evidence, however,
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as to whether this transporter is located in either the canalicular or basolateral membrane [13,14].
A P-type ATPase in the canalicular membrane Not all hepatic transporters that underlie liver disease are ABC proteins. The defects underlying two forms of progressive familial intrahepatic cholestasis have already been discussed. A third form, with a low γGT, has been shown to be caused by mutations in the FIC1 (familial intrahepatic cholestasis 1) gene [15]. The same gene is also mutated in the milder phenotype — benign recurrent intrahepatic cholestasis. FIC1 is a member of the third subfamily of P-type ATPases [16]. Members of this subfamily are probably aminophospholipid transporters, responsible for maintaining the enrichment of phosphatidylserine and phosphatidylethanolamine on the inner leaflet of the plasma membrane in comparison to the outer leaflet. It remains very unclear how a defect in this cellular function can lead to cholestasis with low levels of bile acids in bile. The expression of FIC1, however, is not restricted to the liver [15]; there are higher levels of expression in the upper gastrointestinal tract and pancreas. This correlates with the observation that some of these patients have had pancreatitis and have malabsorbtion which persists after liver transplantation. Recent data, presented in abstract form only, indicates that in the liver this protein is expressed in the canalicular membrane [17].
ABC1 has a key role in cholesterol efflux An ABC transporter of huge potential significance has recently been identified as being mutated in a human disease. ABC1 is the product of ABCA1 [18], recessive mutations in which have been identified in patients with Tangier disease [19•–24•]. Additionally, a heterozygous 3 base pair deletion was found in another pedigree, which segregates the dominant trait, familial high-density lipoprotein deficiency (FHA) [22•]. Tangier disease is an extreme form of FHA with other features [25]. In addition to having very low levels of high-density lipoprotein (HDL) in the blood, patients have a marked intracellular accumulation of cholesterol ester. This latter feature is manifested most dramatically as enlarged yellow/orange tonsils and less dramatically as hepatosplenomegaly and as a peripheral neuropathy. The obvious interpretation of these findings is that ABC1 plays a critical role in the HDL-mediated cholesterol efflux from a wide variety of cell types. For this reason, one group have renamed the protein ‘cholesterol efflux regulatory protein’ [21•,22•]. The function of the protein in this critical process is further supported by a reversible reduction in cholesterol efflux from cultured cells after transfection with ABCA1 antisense oligonucleotides [19•–22•]. It should be noted that although ABC1 has a critical role in cholesterol efflux, it has not been demonstrated to be a cholesterol transporter and its subcellular location has not been determined. It is also of interest that
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the closest human homologue to ABCA1 is ABCA4 (previously known as ABCR), which encodes the Rim protein, found in ocular photoreceptors and which is mutated in Stargardt’s disease [26–28]. The molecular function of the Rim protein has not been determined but the fact that the intracellular accumulations in Tangier disease include carotenoids may point to some similarity of function. Most importantly, however, low levels of HDL–cholesterol are associated with premature atherosclerotic arterial disease, and it will be very interesting to see if ABC1 abnormalities contribute to this much more common phenotype.
HHH syndrome reveals a mitochondrial transporter Many diseases have been shown to be caused by defects in intracellular transport, usually into or out of subcellular organelles. An important recent example is the identification of the genetic basis of hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome [29••]. The four enzymatic steps required to complete the urea cycle have been understood for many years but three of these steps occur in the cytosol whilst ornithine carbamyltransferase is situated in the mitochondria. It was suggested as long ago as 1974 [30] that the biochemical findings present in HHH syndrome could all be attributed to a defect in the transport of ornithine across the mitochondrial membrane. Although ornithine is conserved in the urea cycle, there is a general need for ornithine degradation, this process also occurs within the mitochondrial matrix. The authors of the recent paper used the sequences of known ornithine transporters, from fungi, to search the ‘dbEST’ database of expressed sequence tags. Only ESTs with one particular conserved sequence were considered. In addition, they selected for transcripts which showed increased expression after protein loading. Only one EST was highly expressed in the liver and met these criteria. Three other overlapping clones were required to complete the 2640 bp cDNA sequence. Mutations were found in patients and partial restoration of ornithine incorporation was restored to cultured fibroblasts from an HHH syndrome patient, following transfection with the recombinant gene. The gene has been termed ORNT1. This elegant piece of work is in part testament to the amount of data that is rapidly accumulating in genetic databases. Indeed a quick search of the public genomic sequencing databases reveals that part of the gene is already represented in a sequence tag connector [31] and that there are almost certainly homologous genes on chromosomes 5 and 22.
Conclusions It is not clear into which diagnostic category most of these diseases should be placed. They are not metabolic diseases in so much as the defect is not primarily one of metabolism. Many present, however, in the same way as metabolic diseases — HHH syndrome being a good example. Classic metabolic diseases can be characterised by the identification
of accumulated precursor molecules or by the absence of metabolites. By contrast, however, many transporter defects are not manifested by abnormal serum levels of their substrate. Even in those defects where there is a good correlation between the biochemical phenotype and the molecular defect, any assay of function would usually have to be performed with whole cells or cellular fractions. This has rendered them much less amenable to classic biochemical analysis. Molecular genetics has no such handicap. Indeed linkage analysis is the ideal tool for localisation and subsequent identification of genes mutated in disease when the underlying biochemical defect is not understood. In some cases, such as the MDR3 gene (ABCB4), even when the gene has been expressed in vitro, the function is still not clear. In these cases, animal ‘knockout’ models have been required to elucidate the true function. Thus a combination of molecular genetic approaches: linkage analysis, expression cloning, animal models and the use of genome project data has lead to a rapid increase in our understanding of cellular transport processes and of the diseases associated with them.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1.
Juliano RL, Ling V: A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976, 455:152-162.
2.
Smit JJ, Schinkel AH, Oude Elferink RP, Groen AK, Wagenaar E, van Deemter L, Mol CA, Ottenhoff R, van der Lugt NM, van Roon MA et al.: Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell 1993, 75:451-462.
3.
van der Bliek AM, Kooiman PM, Schneider C, Borst P: Sequence of mdr3 cDNA encoding a human P-glycoprotein. Gene 1988, 71:401-411.
4.
Deleuze JF, Jacquemin E, Dubuisson C, Cresteil D, Dumont M, Erlinger S, Bernard O, Hadchouel M: Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996, 23:904-908.
5.
de Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, Deleuze JF, Desrochers M, Burdelski M, Bernard O et al.: Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 1998, 95:282-287.
6.
Jacquemin E, Cresteil D, Manouvrier S, Boute O, Hadchouel M: Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. Lancet 1999, 353:210-211.
7.
Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, Hofmann AF, Meier PJ: The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998, 273:10046-10050.
8.
Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, Arnell H, Sokal E, Dahan K, Childs S, Ling V et al.: A gene encoding a liverspecific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet 1998, 20:233-238.
9.
Strautnieks SS, Kagalwalla AF, Tanner MS, Knisely AS, Bull L, Freimer N, Kocoshis SA, Gardiner RM, Thompson RJ: Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24. Am J Hum Genet 1997, 61:630-633.
10. Jansen PL, Strautnieks SS, Jacquemin E, Hadchouel M, Sokal EM, • Hooiveld GJ, Koning JH, De Jager-Krikken A, Kuipers F, Stellaard F et al.: Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis. Gastroenterology 1999, 117:1370-1379. Further evidence of the phenotypic features, with genetic correlation, seen in bile salt export pump deficiency.
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11. Paulusma CC, Kool M, Bosma PJ, Scheffer GL, ter Borg F, Scheper RJ, Tytgat GN, Borst P, Baas F, Oude Elferink RP: A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology 1997, 25:1539-1542.
22. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van • Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO et al.: Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999, 22:336-345. See annotation [24•].
12. Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, Scheper RJ, Borst P, Oude Elferink RP: Congenital jaundice in rats with a mutation in a multidrug resistanceassociated protein gene. Science 1996, 271:1126-1128.
23. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, • Brewer HB, Duverger N, Denefle P et al.: Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999, 22:352-355. See annotation [24•].
13. Ortiz DF, Li S, Iyer R, Zhang X, Novikoff P, Arias IM: MRP3, a new ATP-binding cassette protein localized to the canalicular domain of the hepatocyte. Am J Physiol 1999, 276:G1493-G1500. 14. Konig J, Rost D, Cui Y, Keppler D: Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 1999, 29:1156-1163. 15. Bull LN, van Eijk MJ, Pawlikowska L, DeYoung JA, Juijn JA, Liao M, Klomp LW, Lomri N, Berger R, Scharschmidt BF et al.: A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 1998, 18:219-224. 16. Tang X, Halleck MS, Schlegel RA, Williamson P: A subfamily of P-type ATPases with aminophospholipid transporting activity. Science 1996, 272:1495-1497. 17.
Ujhazy P, Ortiz DF, Misra S, Li S, Arias IM: ATP-dependent aminophospholipid translocase activity in rat canalicular membrane vesicles and its relationship to FIC1. Hepatology 1999, 30:462A.
18. Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G, Kaminski WE, Schmitz G: Molecular cloning of the human ATPbinding cassette transporter 1 (hABC1): evidence for steroldependent regulation in macrophages. Biochem Biophys Res Commun 1999, 257:29-33. 19. Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, • Seilhamer JJ, Vaughan AM, Oram JF: The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest 1999, 104:R25-R31. See annotation [24•]. 20. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, • Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M et al.: The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999, 22:347-351. See annotation [24•]. 21. Marcil M, Brooks-Wilson A, Clee SM, Roomp K, Zhang LH, Yu L, • Collins JA, van Dam M, Molhuizen HO, Loubster O et al.: Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux. Lancet 1999, 354:1341-1346. See annotation [24•].
24. Remaley AT, Rust S, Rosier M, Knapper C, Naudin L, Broccardo C, • Peterson KM, Koch C, Arnould I, Prades C et al.: Human ATPbinding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original tangier disease kindred. Proc Natl Acad Sci USA 1999, 96:12685-12690. This and the preceding papers [19•–23•] contain the same basic message: mutations in the gene encoding ABC1 lead to a failure of cholesterol export from cells into HDL. This is potentially of huge physiological and medical significance. 25. Fredrickson DS, Altrocchi PH, Avioli LV, Goodman DS, Goodman HC: Tangier disease — combined clinical staff conference at the National Institutes of Health. Ann Intern Med 1961, 55:1061. 26. Allikmets R, Shroyer NF, Singh N, Seddon JM, Lewis RA, Bernstein PS, Peiffer A, Zabriskie NA, Li Y, Hutchinson A et al.: Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration. Science 1997, 277:1805-1807. 27.
Allikmets R, Singh N, Sun H, Shroyer NF, Hutchinson A, Chidambaram A, Gerrard B, Baird L, Stauffer D, Peiffer A et al.: A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet 1997, 15:236-246.
28. Azarian SM, Travis GH: The photoreceptor rim protein is an ABC transporter encoded by the gene for recessive Stargardt’s disease (ABCR). FEBS Lett 1997, 409:247-252. 29. Camacho JA, Obie C, Biery B, Goodman BK, Hu CA, Almashanu S, •• Steel G, Casey R, Lambert M, Mitchell GA et al.: Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter. Nat Genet 1999, 22:151-158. In this study of HHH syndrome, significantly aided by components of the human genome project, a gene defect and a gene product are identified. 30. Fell V, Pollitt RJ, Sampson GA, Wright T: Ornithinemia, hyperammonemia, and homocitrullinuria. A disease associated with mental retardation and possibly caused by defective mitochondrial transport. Am J Dis Child 1974, 127:752-756. 31. Mahairas GG, Wallace JC, Smith K, Swartzell S, Holzman T, Keller A, Shaker R, Furlong J, Young J, Zhao S et al.: Sequence-tagged connectors: a sequence approach to mapping and scanning the human genome. Proc Natl Acad Sci USA 1999, 96:9739-9744.
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Inherited disorders of transport in the liver Richard Thompson* and Sandra Strautnieks There has been a recent explosion in our understanding of hepatic transport processes. Much of this has resulted from the investigation of human diseases involving the liver and the use of animal models. The physiological roles of many of these transporters have been well characterised previously but have, until now, been resistant to molecular cloning. Addresses Department of Child Health, Guy’s, King’s and St Thomas’ School of Medicine, King’s College Hospital, London, SE5 9RS, UK *e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:310–313 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations ABC ATP-binding cassette BSEP bile salt export pump cMOAT canalicular multi-specific organic anion transporter HDL high-density lipoprotein HHH hyperornithinemia–hyperammonemia–homocitrullinuria MDR multidrug resistance MRP MDR-associated protein PFIC progressive familial intrahepatic cholestasis PHHI persistent hyperinsulinemia hypoglycemia of infancy
Introduction The adult human liver weighs ~1.5 kg and has a blood flow of ~1.5 litres per minute. 80% of the blood flow occurs via the hepatic portal vein. This key aspect of hepatic anatomy is critical to many of its functions. The gastrointestinal tract is our largest source of exposure to exogenous compounds. Some of these substances are potentially harmful though many are, of course, nutrients. In the form in which they are absorbed from the gastrointestinal tract, however, even many nutrients are often not beneficial to most cells in the body. Furthermore, most cells in the body require a steady supply of substrates rather than surges following meals. The function of the liver in this respect is to remove from the blood a large proportion of what is absorbed from the gut prior to the blood circulating to the rest of the body. In order to achieve this function, a multitude of transporter polypeptides are expressed in the hepatocyte basolateral membrane. The liver also has an excretory function: bile is formed by a separate set of transporter molecules in the hepatocyte canalicular membrane. The main purpose of bile production would appear to be the need to excrete bile acids into the gastrointestinal tract to the liver to absorb fats efficiently. Most other biliary constituents are either protective or could be excreted, though with less efficiency, via another route such as the kidney. In addition to uptake and efflux mechanisms, an increasing number of intracellular transporters have been identified and their genes cloned — many of which are not hepatocyte-specific.
Many of the molecules underlying a number of key hepatic transport processes have eluded cloning and characterisation attempts until recently. The study of patients with apparent defects of hepatic transport, however, has recently advanced our knowledge greatly. This has been aided further by the quantity and quality of information being generated by the human genome project. Some of the important transport processes within the liver that have been elucidated in this way are reviewed here.
The ABC of hepatocyte canalicular transporters Mutations in four human canalicular transporters have been shown to underlie different human diseases. We discuss these later. However there seems to be a much greater redundancy in the basolateral transporters. Not only do most basolateral transporters have a wide substrate specificity but there also is a good deal of overlap. The consequence is that no human disease has yet been associated with a single hepatocyte-specific basolateral transporter. Four key canalicular transporters are members of the ATPbinding cassette (ABC) superfamily. Three are even more closely related and are members of the ABCB family, which until recently was known as the MDR (multidrug resistance) family. The original member of the family was MDR1, also known as P-glycoprotein [1]. The name for the gene encoding this protein has recently changed from PGY1 to ABCB1. The gene is located on chromosome 7q21.1 somewhat proximal to the best-known ABC transporter encoding gene — CFTR. As the name suggests, this protein was identified as a result of its ability to confer resistance to chemotherapy agents in tumour cells. In fact this function is not an epiphenomenon but probably derives from its true physiological function. It is capable of transporting a wide range of drugs. No human disease has so far been attributed to mutations in this gene, and the ‘knockout’ mouse is essentially normal [2]. However, drug handling in the murine model was far from normal both with respect to renal and hepatic excretion and also with respect to central nervous system penetration. MDR1 is certainly expressed in the blood–brain barrier. Polymorphic variations in ABCB1 probably confer variation in drug distribution and elimination in man. ABCB4 encodes MDR3 [3]. ABCB4 was known as PGY3 and the mouse gene product is confusingly known as mdr2. The human gene is also found on 7q21.1 close to ABCB1. Homozygous knockout mice for this gene were recognised to lack biliary phospholipid excretion [2]. It was subsequently realised that a group of patients with a severe form of liver disease had histological features similar to the knockout mice [4]. This disease has become known as ‘high γGT progressive familial intrahepatic cholestasis’ (high γGT PFIC). Both reduced levels of ABCB4 mRNA,
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and (subsequently) coding sequence mutations have been found in these patients [5]. As with the knockout mice they also either have dramatically reduced or absent biliary phospholipid excretion. Importantly, one of the mothers — an obligate heterozygote for an ABCB4 mutation — had cholestasis during pregnancy [5]. Subsequently, a large family has been reported in which four obligate heterozygote mothers and two other relatives had cholestasis during pregnancy [6]. The third member of the MDR family expressed in the canalicular membrane is the bile salt export pump (BSEP). When first identified the gene was termed ‘sister of P-glycoprotein’, it was then termed BSEP but has now been designated ABCB11. This gene was originally identified as a pig expressed sequence tag (EST), and subsequently cloned in rat [7] and human [8]. The later was undertaken after a form of low γGT PFIC was mapped by linkage analysis to the same locus [9]. These patients have very low levels of bile acids in their bile, despite normal bile acid synthesis and high levels in serum [10•]. The rat orthologue has been expressed in vitro and is a competent bile acid transporter with a Km of the order to be expected from previous physiology studies using canalicular membrane vesicles [7]. It has also been shown recently that there is a good correlation between mutations in ABCB11 and a lack of BSEP-specific immuno-staining of liver. Several patients also had biliary bile acid analysis performed, confirming the near total absence of excretion [10•]. The fourth ABC transporter present in the hepatocyte canalicular membrane is the canalicular multi-specific organic anion transporter (cMOAT). Again the gene name has recently changed, from CMOAT to ABCC2. The C family of ABC transporters are more heterogeneous in structure than the B family and include the cystic fibrosis transmembrane conductance regulator (encoded by ABCC7) and the sulfonylurea receptor (encoded by ABCC8). The latter is mutated in some cases of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). CMOAT is also known as multidrug-resistance-associated protein 2 (MRP2). It is not clear whether it is capable of conferring drug resistance but it does transport a wide range of conjugated anions across the canalicular membrane. The major endogenous substrates are bilirubin glucuronides. Autosomal recessive mutations have been identified in this gene in patients with Dubin–Johnson syndrome [11]. This mild disease is characterised by conjugated hyperbilirubinemia and the deposition of very dark pigment in the liver. The pigment is not bilirubin. In both Dubin–Johnson syndrome patients and the TRmutant and Eisai hyperbilrubinemic rat models [12], there is overexpression of another ABC transporter — MRP3 [13,14], which is encoded by ABCC3. It has been suggested that this overexpression is a compensatory mechanism allowing bilirubin efflux, though clearly not of the pigment that accumulates. There is conflicting evidence, however,
311
as to whether this transporter is located in either the canalicular or basolateral membrane [13,14].
A P-type ATPase in the canalicular membrane Not all hepatic transporters that underlie liver disease are ABC proteins. The defects underlying two forms of progressive familial intrahepatic cholestasis have already been discussed. A third form, with a low γGT, has been shown to be caused by mutations in the FIC1 (familial intrahepatic cholestasis 1) gene [15]. The same gene is also mutated in the milder phenotype — benign recurrent intrahepatic cholestasis. FIC1 is a member of the third subfamily of P-type ATPases [16]. Members of this subfamily are probably aminophospholipid transporters, responsible for maintaining the enrichment of phosphatidylserine and phosphatidylethanolamine on the inner leaflet of the plasma membrane in comparison to the outer leaflet. It remains very unclear how a defect in this cellular function can lead to cholestasis with low levels of bile acids in bile. The expression of FIC1, however, is not restricted to the liver [15]; there are higher levels of expression in the upper gastrointestinal tract and pancreas. This correlates with the observation that some of these patients have had pancreatitis and have malabsorbtion which persists after liver transplantation. Recent data, presented in abstract form only, indicates that in the liver this protein is expressed in the canalicular membrane [17].
ABC1 has a key role in cholesterol efflux An ABC transporter of huge potential significance has recently been identified as being mutated in a human disease. ABC1 is the product of ABCA1 [18], recessive mutations in which have been identified in patients with Tangier disease [19•–24•]. Additionally, a heterozygous 3 base pair deletion was found in another pedigree, which segregates the dominant trait, familial high-density lipoprotein deficiency (FHA) [22•]. Tangier disease is an extreme form of FHA with other features [25]. In addition to having very low levels of high-density lipoprotein (HDL) in the blood, patients have a marked intracellular accumulation of cholesterol ester. This latter feature is manifested most dramatically as enlarged yellow/orange tonsils and less dramatically as hepatosplenomegaly and as a peripheral neuropathy. The obvious interpretation of these findings is that ABC1 plays a critical role in the HDL-mediated cholesterol efflux from a wide variety of cell types. For this reason, one group have renamed the protein ‘cholesterol efflux regulatory protein’ [21•,22•]. The function of the protein in this critical process is further supported by a reversible reduction in cholesterol efflux from cultured cells after transfection with ABCA1 antisense oligonucleotides [19•–22•]. It should be noted that although ABC1 has a critical role in cholesterol efflux, it has not been demonstrated to be a cholesterol transporter and its subcellular location has not been determined. It is also of interest that
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the closest human homologue to ABCA1 is ABCA4 (previously known as ABCR), which encodes the Rim protein, found in ocular photoreceptors and which is mutated in Stargardt’s disease [26–28]. The molecular function of the Rim protein has not been determined but the fact that the intracellular accumulations in Tangier disease include carotenoids may point to some similarity of function. Most importantly, however, low levels of HDL–cholesterol are associated with premature atherosclerotic arterial disease, and it will be very interesting to see if ABC1 abnormalities contribute to this much more common phenotype.
HHH syndrome reveals a mitochondrial transporter Many diseases have been shown to be caused by defects in intracellular transport, usually into or out of subcellular organelles. An important recent example is the identification of the genetic basis of hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome [29••]. The four enzymatic steps required to complete the urea cycle have been understood for many years but three of these steps occur in the cytosol whilst ornithine carbamyltransferase is situated in the mitochondria. It was suggested as long ago as 1974 [30] that the biochemical findings present in HHH syndrome could all be attributed to a defect in the transport of ornithine across the mitochondrial membrane. Although ornithine is conserved in the urea cycle, there is a general need for ornithine degradation, this process also occurs within the mitochondrial matrix. The authors of the recent paper used the sequences of known ornithine transporters, from fungi, to search the ‘dbEST’ database of expressed sequence tags. Only ESTs with one particular conserved sequence were considered. In addition, they selected for transcripts which showed increased expression after protein loading. Only one EST was highly expressed in the liver and met these criteria. Three other overlapping clones were required to complete the 2640 bp cDNA sequence. Mutations were found in patients and partial restoration of ornithine incorporation was restored to cultured fibroblasts from an HHH syndrome patient, following transfection with the recombinant gene. The gene has been termed ORNT1. This elegant piece of work is in part testament to the amount of data that is rapidly accumulating in genetic databases. Indeed a quick search of the public genomic sequencing databases reveals that part of the gene is already represented in a sequence tag connector [31] and that there are almost certainly homologous genes on chromosomes 5 and 22.
Conclusions It is not clear into which diagnostic category most of these diseases should be placed. They are not metabolic diseases in so much as the defect is not primarily one of metabolism. Many present, however, in the same way as metabolic diseases — HHH syndrome being a good example. Classic metabolic diseases can be characterised by the identification
of accumulated precursor molecules or by the absence of metabolites. By contrast, however, many transporter defects are not manifested by abnormal serum levels of their substrate. Even in those defects where there is a good correlation between the biochemical phenotype and the molecular defect, any assay of function would usually have to be performed with whole cells or cellular fractions. This has rendered them much less amenable to classic biochemical analysis. Molecular genetics has no such handicap. Indeed linkage analysis is the ideal tool for localisation and subsequent identification of genes mutated in disease when the underlying biochemical defect is not understood. In some cases, such as the MDR3 gene (ABCB4), even when the gene has been expressed in vitro, the function is still not clear. In these cases, animal ‘knockout’ models have been required to elucidate the true function. Thus a combination of molecular genetic approaches: linkage analysis, expression cloning, animal models and the use of genome project data has lead to a rapid increase in our understanding of cellular transport processes and of the diseases associated with them.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1.
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