In the past decade many new transporters have been SPECIAL REPORTS AND REVIEWS

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1 GASTROENTEROLOGY 2006;130: SPECIAL REPORTS AND REVIEWS Hepatocanalicular Transport Defects: Pathophysiologic Mechanisms of Rare Diseases RONALD P. J. OUDE ELFERINK, COEN C. PAULUSMA, and ALBERT K. GROEN AMC Liver Center, Academic Medical Center, Amsterdam, The Netherlands See CME Quiz on page 931. The apical membrane of the hepatocyte fulfils a unique function in the formation of primary bile. For all important biliary constituents a primary active transporter is present that extrudes or translocates its substrate toward the canalicular lumen. Most of these transporters are ATP-binding cassette (ABC) transporters. Two types of transporters can be recognized: those having endogenous metabolites as substrates (which could be referred to as physiologic transporters) and those involved in the elimination of drugs, toxins, and waste products. It should be emphasized that this distinction cannot be strictly made as some endogenous metabolites can be regarded as toxins as well. The importance of the canalicular transporters has been recognized by the pathologic consequence of their genetic defects. For each of the physiologic transporter genes an inherited disease has now been identified and most of these diseases have a quite serious clinical phenotype. Strikingly, complete defects in drug transporter function have not been recognized (yet) or only cause a mild phenotype. In this review we only briefly discuss the inherited defects in transporter function, and we focus on the pathophysiologic concepts that these diseases have generated. In the past decade many new transporters have been identified, including a large set of transporters relevant to liver physiology. Initially, these transporters as well as their genes have been given trivial names giving rise to confusion. More recently, a standardized nomenclature was designed which should take away much of this confusion. The same holds for diseases that are associated with mutations in these genes. To provide clarity, we indicate both the standard and trivial names of transporter genes and of diseases in Table 1. The genes, gene products, their (putative) functions, and the corresponding diseases are also depicted in Figure 1. At the end of this review, an appendix glossary is added that explains many of the terms and acronyms used in this review. Furthermore, we adhere to the standard annotation of genes and proteins; reference to genes is made by writing the code in italics, whereas proteins are referred to by writing the code in normal case. When a human gene is indicated all letters are in capitals, whereas genes and proteins from all other species are in lower case (except the first letter). As an example, the human gene is ATP8B1, the protein derived from this gene (gene product) is referred to as ATP8B1; in mice or any other nonhuman species these are Atp8b1 and Atp8b1, respectively. Inherited Canalicular Transport Defects A group of very rare inherited forms of cholestasis has been defined in the last decade by linking genetic analysis to clinical phenotype. These disorders are now referred to as progressive familial intrahepatic cholestasis (PFIC). Three subgroups of patients (PFIC type 1 3) can be defined on the basis of mutations in the genes for 3 relevant canalicular transporter genes. 1 The background of this somewhat confusing nomenclature is that the clinical phenotype was considered to be very similar for a long time. Upon genetic distinction of the three types of PFIC, subtle differences in the clinical presentation have become more evident. It must be mentioned here that a fourth group of patients is often referred to as PFIC type 4. These include patients with defects in the bile salt synthetic pathway. There is a rationale for inclusion of this subgroup of patients as the clinical phenotype of this group of rare disorders is very similar to PFIC types 1 and 2. These Abbreviations used in this paper: ABC, ATP-binding cassette; BRIC, benign recurrent intrahepatic cholestasis; PFIC, progressive familial intrahepatic cholestasis; POPC, palmitoyl-oleoyl-pc; P-SM, palmitoyl- SM; PS, phosphatidylserine by the American Gastroenterological Association Institute /06/$32.00 doi: /j.gastro

2 March 2006 HEPATOCANALICULAR TRANSPORT DEFECTS 909 Table 1. Genes and Genetic Defects in Canalicular Transport Disease Symptoms Laboratory findings Mutated gene (trivial name gene product) Function gene product PFIC type 1 (Bylers disease) Cholestasis, diarrhea Low serum GGT ATP8B1 (FICI) Aminophospholipid flippase BRIC type 1 (benign recurrent intrahepatic cholestasis) Periodic attacks of cholestasis Low serum GGT ATP8B1 (FICI) Aminophospholipid flippase PFIC type 2 Cholestasis Low serum GGT ABCB11 (BSEP) Bile salt export pump BRIC type 2 Periodic attacks of Low serum GGT ABCB11 (BSEP) Bile salt excretion cholestasis PFIC type 3 Cholestasis High serum GGT ABCB4 (MDR3) (in rodents: Mdr2) Phosphatidylcholine floppase LPAC syndrome (low phospholipid associated cholelithiasis) Sitosterolemia Intrahepatic gallstones and/or biliary cirrhosis Anemia, premature atherosclerosis High GGT ABCB4 (MDR3) Phosphatidylcholine floppase Accumulation of plant sterols in serum and tissues, hypercholesterolemia Dubin Johnson syndrome Jaundice Conjugated hyperbilirubinemia Intrahepatic cholestasis of Cholestasis, Pruritus Low or high GGT pregnancy (ICP) in the third trimester of pregnancy ABCG5 or ABCG8 (sterolin 1 and 2) ABCC2 (MRP2)? in a minority of cases: ATP8B1 ABCB4 Cholesterol floppase Organic anion pump? Aminophospholipid flippase, phosphatidylcholine floppase patients differ, however, by the fact that pruritus is generally not evident, most likely owing to the absence of normal bile salts. These defects fall beyond the scope of this review. Also other genetic defects associated with cholestasis, such as biliary atresia, Alagille syndrome, arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome, and familial hypercholanemia, are not directly caused by a deficiency in canalicular transporters and therefore are not discussed in this review. Finally, we discuss 2 other genetic diseases caused by mutations in canalicular transporter genes, sitosterolemia and the Dubin Johnson syndrome. PFIC Type 1 and BRIC1. PFIC1 was formerly called Bylers disease after the Amish family in which this disease was first described. 2 Patients usually present at young age (neonatal period) with typical cholestatic symptoms; serum bile salt, bilirubin, and transaminase levels are elevated but, in contrast to other pediatric cholestatic syndromes such as biliary atresia or Alagille syndrome, serum -glutamyltransferase (GGT) levels are low. Liver histology reveals fibrosis but no bile duct proliferation. The patients often develop end-stage liver disease requiring liver transplantation. Electron microscopic analysis of liver tissue, if available, allows a differential diagnosis between PFIC1 and other forms of cholestasis with low serum GGT levels. In PFIC1 liver specimens, the canalicular lumen contains coarse granular bile, whereas in biopsies from livers with other causes of cholestasis, the canalicular lumen contains amorphous bile. 3 The background of this morphologic difference is presently unclear. PFIC1 is the least well understood of the 3 forms of this disease. It is caused by mutations in the ATP8B1 gene. 4 Importantly, mutations in this gene not only cause PFIC1 but also benign recurrent intrahepatic cholestasis (BRIC) type 1. BRIC1 is characterized by episodes of cholestasis, the onset and resolution of which are caused by unknown factors. During cholestatic periods serum bile salt and bilirubin are increased and patients characteristically suffer from pruritus. With the increasing number of genetically identified patients with either of the 2 clinical diagnoses, it has become clear that the 2 syndromes actually represent a continuum in which PFIC1 is the severe end of the spectrum and BRIC the milder form. Indeed, patients who initially were diagnosed as having BRIC may develop a continuous and progressive form of cholestasis that is indistinguishable from PFIC1. 5 Missense mutations are more often associated with BRIC than with PFIC1, and the reverse is true

3 910 OUDE ELFERINK ET AL GASTROENTEROLOGY Vol. 130, No. 3 Figure 1. Inherited diseases associated with mutations in canalicular transporter genes. The figure gives the name of a canalicular transporter gene in bold and the trivial name of its gene product between parentheses. The second line indicates its function and the third line indicates in bold the corresponding disease. for nonsense mutations and deletions. 4 This allows the suggestion that in BRIC patients residual ATP8B1 (FIC1) protein activity may be present, whereas the mutations in PFIC1 patients generally knock out the entire activity. Importantly, in addition to cholestasis, patients with PFIC1 may also suffer from extrahepatic symptoms, such as diarrhea, pancreatitis, hearing problems, and elevated sweat chloride concentrations (the last symptom being similar to what is observed in patients with cystic fibrosis). Liver transplantation resolves the cholestatic phenotype, but extrahepatic symptoms such as diarrhea and failure to thrive remain. 6 Like PFIC1 patients, BRIC patients may develop pancreatitis. 7 More than 15 years ago Whitington et al 8,9 performed chronic partial biliary diversion in low-ggt PFIC patients, which improved the clinical symptoms. Several centers have now adopted this strategy and it turns out to be beneficial in both PFIC1 and PFIC2 (low-ggt PFIC) PFIC Type 2 and BRIC2. PFIC2 is caused by mutations in the ABCB11 gene, which encodes the main canalicular bile salt transporter ABCB11 (BSEP). 13 Liver histology displays portal inflammation and giant-cell hepatitis. Serum bile salt and transaminase activities are increased but GGT is low. As mentioned, PFIC2 can be distinguished from PFIC1 by electron microscopic analysis of liver biopsies, with amorphous bile in the canalicular lumen of PFIC2 liver tissue. 3 Importantly, it was recently reported that patients who are diagnosed for BRIC may have a mutation in the ABCB11 gene rather than in the ATP8B1 gene. 14 Hence, 2 types of BRIC can now be discerned: BRIC1, caused by mutation in ATP8B1, and BRIC2, caused by mutations in ABCB11. The similarity between the 2 syndromes is the episodic cholestasis, but BRIC2 is often associated with gallstones, which is not the case with BRIC1. Conversely, BRIC1 patients may develop pancreatitis and this is not observed in BRIC2 patients. 14 PFIC Type 3. Patients with PFIC3 usually present at a few years of age and suffer from chronic and progressive cholestasis. Liver histology reveals fibrosis (progressing into cirrhosis) with portal inflammation and, in contrast to the other forms of PFIC, strong bile duct proliferation. As a consequence of the cirrhosis, the patients are prone to gastrointestinal bleeding. Another difference with the 2 other forms of PFIC is a characteristic high serum GGT activity. About 50% of the patients need a liver transplantation; the other half may benefit from treatment with UDCA. 15 This type of PFIC is caused by mutations in the ABCB4 (MDR3) gene, which leads to defective biliary phospholipid secretion. Translocation of phosphatidylcholine from the inner leaflet of the canalicular membrane to the outer leaflet is mediated by ABCB4 and makes phospholipid available for extraction by bile salts. 1,16 This process is essential for protection of the canalicular membrane of hepatocytes, as

4 March 2006 HEPATOCANALICULAR TRANSPORT DEFECTS 911 well as the apical membrane of cholangiocytes against the detergent action of bile salts. Biliary bile salt secretion is normal in these patients. The absence of biliary phospholipid excretion together with normal bile salt excretion causes damage to membranes. 15,17 More recently it has become clear that mutations in the ABCB4 gene that may reduce but not eliminate activity of the protein can cause a variety of milder PFIC3 phenotypes. This also applies in case of heterozygosity for mutations that eliminate transporter activity. 18 Mutations in the ABCB4 gene have been detected in patients with symptoms of primary biliary cirrhosis (PBC). 19 Such patients may be primarily if not exclusively found in the subgroup of PBC patients without antimitochondrial antibodies (AMA negative), that is, patients with symptoms of PBC that may not be caused by autoimmunity. Lucena et al 19 as well as Thompson et al 20 reported on AMA-negative patients with mutations in the ABCB4 gene, whereas Pauli-Magnus et al 21 could not find ABCB4 mutations in a group of PBC patients with antimitochondrial antibodies (AMA positive). Furthermore, ABCB4 mutations have been reported in patients with intrahepatic cholesterol gallstones (see below). 19,22 Sitosterolemia. Sitosterolemia is caused by a defect in intestinal and biliary sterol transport. This transport is mediated by the 2 half transporters ABCG5 and ABCG8, which together form a functional sterol transporter Patients with this very rare inherited disease have a mutation in either of the 2 genes and hyperabsorb (plant) sterols in the intestine, leading to increased levels in blood and tissues. The accumulation of plant sterols induces hemolysis and sterol accumulation in the form of tendon and/or tuberous xanthomas. In contrast to other forms of hypercholesterolemia, the serum cholesterol level of patients with sitosterolemia is sensitive to dietary cholesterol intake owing to increased intestinal absorption of sterols. 26 Subsequent to the genetic elucidation of sitosterolemia, mice were produced in which the homologous Abcg5 and/or Abcg8 were disrupted, 27,28 and these animals are a phenocopy of human sitosterolemia; they have strongly increased plasma (plant)sterol levels. In these mice, a strong reduction in biliary cholesterol excretion was observed, indicating that these half transporters are also involved in canalicular cholesterol translocation. 27,28 Dubin Johnson Syndrome. The Dubin Johnson syndrome is caused by mutations in the ABCC2 gene encoding MRP2, a canalicular ABC transporter that extrudes organic anions and neutral compounds. Diagnostically, these patients have a chronic conjugated hyperbilirubinemia without elevated serum GGT or transaminases. 1 This rare syndrome is considered to be benign, although anecdotal reports of primary hepatocellular carcinoma have been published Although the best known substrate is conjugated bilirubin, MRP2 should be considered a drug transporter responsible for elimination of endogenous and exogenous compounds. In many cases this concerns products of phase I and phase II metabolism. Other important drug transporters in the canalicular membrane are MDR1 P-glycoprotein (encoded by the ABCB1 gene), which extrudes amphipathic neutral and cationic compounds and BCRP (encoded by the ABCG2 gene), which eliminates amphipathic neutral and anionic compounds. Polymorphisms in these 2 genes have been reported, but these do not seem to cause clinical symptoms. 32,33 Canalicular Lipid Transport Defects Can Cause Gallstone Formation It is well established that supersaturation of bile with cholesterol, which occurs in a large proportion of humans, leads to the formation of cholesterol gallstones. Biliary cholesterol solubilization depends not only on the concentration of the sterol itself but also on the bile salt and phospholipid concentration. 34 Mixed micelles of bile salts and phospholipids solubilize cholesterol better than simple bile salt micelles. Hence, the rate of phospholipid excretion can be expected to be an important factor in the prevention of gallstone formation. This concept was validated by the finding that Abcb4 / (Mdr2 / ) mice, which have a complete defect in phospholipid excretion, develop cholesterol gallstones. 35 On a normal diet, these mice excrete very little cholesterol into bile, because the endogenous bile salts are insufficiently strong detergents to solubilize cholesterol in the absence of phospholipid. However, when the bile salt pool is largely replaced by taurocholate (upon cholate feeding), these mice start to excrete cholesterol and abundant cholesterol crystal formation is observed. 36 In normal humans, biliary cholesterol saturation is already high. One might therefore expect that also partial defects in phospholipid secretion may cause cholesterol gallstone formation. This has, indeed, been shown. Individuals with mutations in the ABCB4 gene are particularly prone to cholesterol gallstone formation. Gallstone formation was observed in several PFIC type 3 patients 15 and, conversely, mutations in ABCB4 were observed in patients with intrahepatic gallstones. 19,22 Importantly, the latter group of patients were not diagnosed as PFIC patients. Because these patients were adults with symptoms differing from PFIC type 3, it was hypothesized that this concerned a mild form of PFIC caused by mutations in ABCB4 that leave

5 912 OUDE ELFERINK ET AL GASTROENTEROLOGY Vol. 130, No. 3 residual activity of the transporter. Rosmorduc et al 22 designated this as low phospholipids-associated cholelithiasis syndrome. More recently, it was reported that cholelithiasis is also frequently observed in patients with PFIC type 2 37 and in patients with BRIC type 2 14 who have mutations in the ABCB11 gene. An analysis of genetic traits that play a role in cholelithiasis in mice also revealed a potential role for Abcb11. The groups of Carey and Paigen 38 compared gallstone-susceptible C57L mice with resistant AKR mice and found several loci that determine susceptibility to formation of cholesterol gallstones. Interestingly, the Lith1 locus on chromosome 2 harbors the Abcb11 gene, which encodes the canalicular bile salt export pump (Bsep). Unexpectedly, Wang et al 39 observed that the susceptible C57L mice have a higher rather than a lower expression of Abcb11 compared with the resistant AKR mice. In line with this observation Wang et al 39 observed higher bile salt excretion rates in C57L mice. Green et al 40 confirmed the higher Abcb11 mrna level in C57L mice but at the same time found a strongly reduced bile salt transport activity in plasma membrane vesicles from these mice compared with the resistant AKR strain. Hence, it remains to be determined by which mechanism the expression level of ABCB11 determines susceptibility to gallstone formation. Association Between Canalicular Transport Defects and Cholestasis of Pregnancy Intrahepatic cholestasis of pregnancy is a reversible form of cholestasis that may develop in the third trimester of pregnancy and usually rapidly resolves after delivery. The incidence of intrahepatic cholestasis of pregnancy lies between 10 and 100 cases per 10,000 pregnancies, but there is a strong ethnic background to this phenomenon. Notably, in the Chilean population intrahepatic cholestasis of pregnancy develops in as much as 16% of pregnancies and within the Araucanian Indian subpopulation it is as high as 28%. 41 The main symptoms are pruritus and, to a lesser extent, jaundice. Serum bile salt levels are increased. 42,43 Increased incidence of fetal distress, premature birth, and even stillbirth in association with intrahepatic cholestasis of pregnancy have been reported (for a review see Paus et al 44 ). It is generally accepted that women who have suffered from intrahepatic cholestasis of pregnancy are also susceptible to development of cholestasis on the use of oral contraceptives. Several reports have shown that women with mutations in the ABCB4 gene can develop intrahepatic cholestasis of pregnancy. 18,45 47 In all cases the reported mutations were shown not to occur in control populations. More recently, intrahepatic cholestasis of pregnancy has also been associated with mutations in the ATP8B1 gene. 48,49 Indeed, intrahepatic cholestasis of pregnancy patients can be divided in a group with low serum GGT (about 70%) and a group with high serum GGT activity (about 30%). 50 In most cases described, the cholestatic symptoms resolved with delivery. Thus, the observed mutations do not cause transporter deficiencies that are strong enough to induce symptoms of PFIC. Most likely, mutations in intrahepatic cholestasis of pregnancy patients reduce transporter activity partly and only give rise to clinical symptoms during pregnancy. It can therefore be hypothesized that during the third trimester of pregnancy there is a generalized impairment of bile formation. Indeed, not only ABCB4 and ABCB11 function are compromised during pregnancy; also ABCC2 deficiency (Dubin Johnson syndrome) may be unmasked during pregnancy The mechanism of this phenomenon has not been completely revealed. It is thought that estrogens play an important role; it is known that estradiol-glucuronide is excreted into bile and, indeed, treatment of rodents with estrogens induces cholestasis. 54 The groups of Vore and Meier provided results to suggest that estradiol-glucuronide needs to be transported into bile (via Mrp2) before it can exert its inhibitory effect on the bile salt pump Abcb11 (Bsep). 55,56 It remains to be demonstrated, however, whether this mechanism is also operative in pregnant women and whether sufficiently high concentrations of the estrogen-glucuronides build up to cause intracanalicular inhibition of the bile salt transporter. Impaired transport may be aggravated by a relocalization of canalicular transporters such as Mrp2. 57 In addition, the observed down-regulation of the basolateral bile salt transporter Ntcp during pregnancy may contribute to the cholestatic effects at the canalicular pole of the cell. 58 Finally, it must be stressed that the present data suggest that only in a minority of intrahepatic cholestasis of pregnancy patients the manifestations are caused by mutations in canalicular transporter genes Down-regulation of Canalicular Transporters in Other Cholestatic Disorders Patients with sepsis often develop cholestatic symptoms and particularly conjugated hyperbilirubinemia, suggesting that the secretion of bilirubin into bile is compromised. 62 These effects have been mimicked in many studies with rodents by injection of LPS and it was subsequently shown that Mrp2 is strongly down-

6 March 2006 HEPATOCANALICULAR TRANSPORT DEFECTS 913 regulated under these conditions. 63 Nuclear receptors, such as FXR and PXR, play a crucial role as transcription factors in the regulation of expression of most canalicular transporter genes, including ABCC2 (encoding MRP2) and ABCB11 (encoding BSEP) as well as the basolateral bile salt transporter NTCP. 59,64 These nuclear receptors have to form heterodimers with their partner RXR and bind their activating ligand before they can exert transcriptional control. Bile salts are activating ligands of FXR; certain xenobiotics are ligands for PXR. Ghose et al 65 showed that LPS induces nuclear export and reduced heterodimerization of RXR which will lead to downregulation of Mrp2. Down-regulation of Rxr has also been observed during acute-phase response. 66 Hence the expression of both NTCP and ABCB11 may also be prone to down-regulation during sepsis. Studies with rodents have shown that sepsis indeed leads to a dramatic down-regulation of Ntcp expression but has only a moderate effect on Abcb11 expression. The basolateral drug transporters Oatp1 and Mrp3 are also down-regulated during acute-phase response. 67 In addition to downregulation of expression, retraction of canalicular transporters in vesicles close to the canalicular membrane may contribute to reduced canalicular transport activity in various disease states such as inflammation, oxidative stress, and estrogen-induced cholestasis. 68 In humans, a few observations have been made that suggest similar mechanisms of down-regulation as in rodents; Hinoshita et al 69 demonstrated a significant reduction in MRP2 expression in patients with HCV infection that correlated with severity of the disease. In addition Zollner et al 70 observed decreased BSEP and MRP2 protein levels in patients with PBC. Patients receiving TPN are also prone to develop cholestasis, and this holds especially for newborn children. The latter is most likely due to the fact that the biliary excretion machinery is immature in newborns; hence, impaired function has a greater impact in newborns than in adults. The mechanism of TPN-associated cholestasis is unknown, but several theories exist with corresponding evidence in animal models and patients. First, components of the intravenous formulations are thought to have cholestatic effects (especially the lipid components 71 ). Second, the lack of release of gastrointestinal hormones such as CCK may lead to reduced induction of bile formation. 72 Third, increased translocation of LPS across the intestinal wall in the absence of enteral nutrition is thought to impair bile flow by mechanisms described. 73 In a mouse model, Tazuke et al 74 recently showed that Abcb4 expression was reduced in mice on TPN, whereas Abcb11 expression was increased. In humans, de Vree et al 75 showed that the absence of enteral nutrition was found to cause a generalized reduction of bile secretion with a particular decrease in the function of ABCB4. This observation suggests that the absence of enteral nutrition rather than toxicity of the TPN solution is the underlying cause. The preferential decrease of phospholipid excretion compared with bile salt excretion may represent a risk factor for hepatic damage owing to insufficient detoxification of toxic bile salts. Along this same line, Geuken et al 76 reported that on liver transplantation the restoration of bile salt excretion was more rapid than that of phospholipid and this observation correlated with the changes in mrna levels of these transporter genes. This discrepant regulation of canalicular transporter expression may represent a cause of liver damage after transplantation. The present knowledge on the regulation of transporter expression by nuclear hormones should render it possible to design strategies that specifically stimulate transporter gene expression in patients on TPN. Canalicular and Intestinal Transporters Work in Tandem For several of the canalicular transporters, it is clear that their simultaneous expression in the hepatocyte and the enterocyte creates a highly efficient system of keeping unwanted compounds out of the systemic circulation. This concept is most dramatically illustrated by the phenotype of sitosterolemia patients and the corresponding mouse model, the Abcg5/g8 knockout. Plant sterol levels in plasma of men and mice lacking this transporter are fold increased. 77 Plant sterols that diffuse into enterocytes are efficiently pumped back into the gut lumen. As a consequence only 5% of ingested plant sterols are absorbed (as opposed to more than 50% of ingested cholesterol). 78 The small amount of plant sterols that does pass this gatekeeper system enters the liver via portal blood and is efficiently extruded into bile by the same transporter, preventing spillover of significant amounts of plant sterols into systemic blood. Clearly, absence of ABCG5/G8 in both tissues leads to a major increase in systemic plant sterol levels. This concept not only acts for the sterol transport by ABCG5/G8, but in fact for all other drug transporters in the canalicular membrane. ABCC2 (MRP2), ABCG2 (BCRP), and ABCB1 (MDR1 Pgp) are all expressed in hepatocytes and in enterocytes. 79 ABCC2 is primarily expressed in the small intestine, 80 whereas ABCG2 is expressed throughout the gut. 81 A number of studies have shown that in the absence of these transporters there is enhanced absorption of drugs and toxins in the gut, leading to enhanced systemic concentrations of drugs and toxins

7 914 OUDE ELFERINK ET AL GASTROENTEROLOGY Vol. 130, No. 3 Figure 2. The different types of lipid translocators in the canalicular membrane of the hepatocyte. Whereas MDR3 P-glycoprotein (ABCB4) and ABCG5/G8 mediate outward translocation ( flopping ) of phosphatidylcholine (PC) and cholesterol, respectively, FIC1 (ATP8B1) is assumed to mediate inward translocation ( flipping ) of aminophospholipids (PS and phosphatidylethanolamine) and possibly also phosphatidylcholine (PC). Molecular Background of Progressive Familial Intrahepatic Cholestasis Type 1 PFIC1 is caused by mutations in the ATP8B1 gene, which encodes a P-type ATPase (also called FIC1). 4 High expression of this gene is observed in pancreas, intestine, and gallbladder, whereas expression in hepatocytes is relatively low. Given the strong expression in nonhepatic tissue, the extrahepatic symptoms in PFIC1 patients may not come as a surprise. On the basis of its sequence, ATP8B1 represents a transporter belonging to the type 4 subfamily of P-type ATPases. Transporters from this subfamily are considered to be aminophospholipid flippases. Thus, they are thought to translocate phospholipids, notably phosphatidylserine (PS) and phosphatidylethanolamine, from the outer to the inner leaflet of the plasma membrane (Figure 2). For ATP8B1 the evidence for such a function is still relatively weak. Ujhazy et al 85 transfected CHO cells with ATP8B1 cdna and observed increased PS translocation as compared with control cells. However, in this study it was not determined whether this difference was significant and it was also not determined whether the protein was localized in the plasma membrane. The closest homologs of the ATP8B1 gene in yeast have been well studied and evidence for a function of the gene product in the maintenance of phospholipid asymmetry is stronger in these cases. 86 Why absence of ATP8B1, if indeed a phospholipid flippase, induces cholestasis remains an open question. PFIC1 was first diagnosed in the Amish Byler family and these patients have a missense mutation resulting in a glycine to valine substitution at position 308 (G308V), the glycine residue being conserved within the type 4 P-type ATPase subfamily. A knock-in mouse model bearing this mutation was generated (the Atp8b1 G308V/G308V mouse) and it could be demonstrated that this mutation leads to near-complete loss of the protein in liver and intestine (and most likely in other tissues of Atp8b1 expression as well). 87 Unfortunately, these mice develop only an extremely mild phenocopy of the human disorder, with very slight (but significant) increases in serum bile salt levels. When these animals are fed a bile salt supplemented diet, they do accumulate massive amounts of bile salt in their circulation. Upon bile salt feeding the animals have increased liver weights and elevated serum transaminase and bilirubin levels and they lose body weight, indicating that under this condition they suffer from liver damage. However, impaired canalicular bile salt secretion could not yet be demonstrated. 87 Most likely, this strikingly different phenotype in the mouse compared to human patients is caused by the strong differences in bile salt metabolism in mice and men. The bile salt composition in humans is considerably more hydrophobic with cholate, chenodeoxycholate (CDCA), and deoxycholate as the main types of bile salts. The last 2 bile salts are dihydroxy bile salts that are more cytotoxic and stronger detergents than those found in mice (mainly muricholate and cholate). 88 Owing to the striking capacity of rodents to (re)hydroxylate secondary bile salts that are generated and absorbed in the intestine, these animals never accumulate substantial amounts of secondary (dihydroxy and monohydroxy) bile salts. Hence, ATP8B1 may play a role in protection of hepatocytes and cholangiocytes against cholestasis induced by these bile salts. Apparently, ATP8B1 is not of direct importance for the function of ABCB11, the canalicular bile salt transporter BSEP; Harris et al 89 have constructed polarized MDCK-II cells that express the bile salt uptake transporter, NTCP, in the basolateral membrane as well as apical ABCB11 and ATP8B1 (or ABCB11 alone). This system allowed them to study vectorial bile salt transport across these cells and they demonstrated that this transport was as efficient before

8 March 2006 HEPATOCANALICULAR TRANSPORT DEFECTS 915 and after transfection with ATP8B1 cdna. It must be borne in mind, however, that MDCKII cells express endogenous Atp8b1 (C. Paulusma et al, unpublished observation), which may have masked effects of exogenous ATP8B1 expression. Chen et al 90 recently reported that in the intestine of 3 patients diagnosed for PFIC1 there was a striking reduction in the expression of FXR, the nuclear receptor that is activated by bile salts and regulates expression of several key genes in bile salt metabolism and transport. 59 They also observed overexpression of ASBT, the ileal uptake transporter for bile salts, in the intestinal tissue of these patients, which is in line with a proposed transcriptional regulation of this gene by FXR. 91 Furthermore, Alvarez et al 92 observed a 60% reduced expression of FXR in the liver of a patient who was genetically diagnosed for PFIC1. Although these observations are intriguing and suggestive for a role of FXR in the primary defect of PFIC1, the number of patients analyzed is too small to draw firm conclusions. It remains to be determined whether a reduction in the expression of FXR is a specific feature of PFIC patients or whether it occurs in more cholestatic conditions. In the latter case, it would represent a secondary rather than a primary consequence of the absence of ATP8B1 functioning. Indeed, Demeilliers et al 93 recently reported in a preliminary form that down-regulation of FXR may be observed in PFIC1, as well as in PFIC2 and biliary atresia patients. We have also observed partial down-regulation of Fxr in Atp8b1 G308V/G308V mice, but only when bile salt accumulation was induced by bile salt feeding and not under control conditions, 87 suggesting that it may be a secondary consequence of high bile salt loads rather than a primary effect of Atp8b1 malfunctioning. Phospholipid Excretion Into Bile Protects Against Bile Salts Two forms of PFIC, types 1 and 3, are caused by defects in canalicular lipid translocators. Sitosterolemia involves a defect in a third lipid translocator, that of sterols. Apparently, lipid asymmetry of the canalicular membrane, more than in any other membrane, is essential for normal physiology. This is not very surprising because more than any other membrane in the body, the canalicular membrane has to withstand very high detergent concentrations. Bile salts, excreted via ABCB11 (BSEP), reach concentrations in the canaliculus well above the critical micellar concentration. This represents a condition that in principle leads to solubilization of the membranes, followed by immediate cell death. Most probably the hepatocyte protects itself against this threat by 2 mechanisms. One mechanism of protection is the organized excretion of phospholipid to form mixed micelles with bile salts. Addition of phospholipids to simple bile salt micelles reduces the capacity of these micelles to take up more phospholipid from the membrane. This organized phospholipid excretion is mediated by ABCB4 (MDR3) in humans (and the homologous Abcb4 [Mdr2 Pgp] in mice). 1,94 This protein mediates translocation of phosphatidylcholine (PC) from the inner to the outer leaflet (Figure 2), but the mechanism of the subsequent step, that is, extraction from the membrane, is largely unknown. It has been known for many years that phospholipid excretion is driven by micelle-forming bile salts. In the past we have proposed that this involves vesiculation from the outer leaflet of the membrane, induced by local translocation of PC to the outer leaflet combined with destabilization of the membrane by luminal bile salts. 16 Indeed, membrane-adherent vesicles have been detected in careful electron microscopic studies. 95,96 Mechanistically it remains difficult, however, to envision a vesiculation process from a single (outer) leaflet of the membrane; this would invoke highly unstable structures in the membrane. In the presence of a high concentration of bile salts, this might present a rather unfavorable situation. More recent information on canalicular bile formation makes this model less likely (see below). Whichever mechanism is used for the extraction of phospholipids into bile, it has proven to be essential for the protection against bile salts: mice with a disruption of the Abcb4 (Mdr2) gene (Abcb4 / mice) develop progressive liver disease, 97,98 and a more severe form of this disease occurs in patients with PFIC3 in which the ABCB4 (MDR3) gene is mutated. 15,17,99 The Abcb4 / mouse has proven to be a very instructive model for our understanding of the role of phospholipid excretion in protection of the liver. The toxic bile leads to a strong increase in hepatocyte turnover owing to increased apoptosis. 97 More recently, Fickert et al 100 performed an elaborate study on the pathology of Abcb4 / mice and noted that bile duct epithelial cells are also particularly affected by the toxic bile that is excreted by these animals. Disruption of the tight junctions and basement membranes of the small bile ducts leads to bile leakage and a subsequent inflammatory response. Lipid Asymmetry in the Canalicular Membrane Is Also Essential for Protection Against Bile Salts The existence of biliary phospholipid excretion is a relatively young development in evolution. Certain mammalian species, such as the guinea pig, 101 excrete very little phospholipid into bile; fish, such as the little skate, do not excrete any phospholipid at all. 102 Indeed, it has been impossible to find a sequence that is homol-

9 916 OUDE ELFERINK ET AL GASTROENTEROLOGY Vol. 130, No. 3 ogous to ABCB4 in the little skate whereas a homolog of ABCB11 (encoding BSEP) has been cloned from this species. 94 Nevertheless, skate excretes a bile alcohol (scymnolsulphate) that is at least as strong a detergent as taurocholate. 102 Because the little skate does not suffer from chronic liver disease as the Abcb4 / mouse does, there must be a second mechanism of protection against high bile salt concentration in the canalicular lumen. Indeed, even in the absence of canalicular phospholipid excretion (in Abcb4 / mice and PFIC3 patients) the hepatocytes must be largely resistant to the extremely high bile salt concentrations as the damage is limited to a chronically increased cell turnover without acute necrosis. Most likely this second mechanism of protection involves the asymmetric lipid distribution of the membrane, with a high content of sphingomyelin and cholesterol in the outer leaflet. In vitro experiments have demonstrated that the combination of these 2 lipids is the only way by which membranes can be rendered virtually detergent insoluble. 103 Although direct proof that this mechanism is responsible for the detergent resistance of the canalicular membrane is lacking, the observed high sphingomyelin content of these membranes isolated from various species makes this hypothesis plausible Cholesterol Is the Crucial Factor in Resistance of the Canalicular Membrane Against Bile Salts Considerable information on detergent (in)solubility has come from the intense cell biological research on lipid rafts in biological membranes. Rafts are thought to play an important role in membrane protein and lipid trafficking as well as cell signaling. 107 Lipid rafts contain specific membrane proteins such as GPI-anchored proteins and have a high content of sphingolipids and cholesterol. At body temperature, membranes composed of glycerophospholipids (phosphatidylcholine, PS, phosphatidylethanolamine) are in the liquid-disordered phase (also referred to as liquid crystalline), in which the lipids are rather loosely packed. The loose packing is mainly caused by the high content of unsaturated fatty acyl chains in glycerophospholipids. Such lipid bilayers are highly prone to intercalation of detergents and subsequent solubilization. However, addition of sphingomyelin and cholesterol to membranes induces a much more rigid membrane structure than is in the liquid-ordered phase. This phase represents tighter packing of lipids, mainly owing to the long, saturated fatty acyl chains of sphingomyelins, which combine quite well with the flat structure of the cholesterol molecule. The behavior of these constituting lipids leads to partial phase separation of the glycerophospholipids plus cholesterol on the one hand and sphingomyelin and (a larger fraction of) cholesterol on the other hand, the first being in the liquiddisordered phase and the second being in the liquidordered phase. 107,108 These phases coexist in the membrane and are most likely in a dynamic equilibrium. Membranes consisting of lipids forming a liquid-ordered phase were found to be resistant to the detergent Triton X By inference, rafts of similar composition are expected to have the same detergent resistance. The isolation of rafts, as preexisting structures, from membrane by virtue of their detergent resistance is controversial 108 ; it is unlikely that rafts are static structures that can be isolated by this highly perturbing method. Nevertheless, these experiments demonstrate that certain lipid compositions can render the membrane detergent resistant and that this resistance depends on the extent to which the constituting lipids are in the liquid-ordered phase. Using data obtained by different analytic methodologies de Almeida et al 109 constructed a phase diagram for the coexistence of the different phases in artificial membranes composed of various ratios of palmitoyl-oleoyl-pc (POPC), palmitoyl-sm (P-SM), and cholesterol. Analyses of these membranes were performed at 23 C, but extrapolated to a similar phase diagram at 37 C. This diagram (Figure 3) reveals that at physiologic membrane lipid compositions there is indeed coexistence of liquid ordered (l o ) and liquid disordered (l d ) phases. A solid ordered (s o ) phase is recognized as well (also referred to as gel phase), which represents an even more rigid membrane structure, but this phase is not thought to occur in biomembranes. The diagram shows that the most important determinant of the liquid-ordered phase is cholesterol. In membranes consisting of POPC and cholesterol, 50% cholesterol yields a membrane with coexisting l o and l d phases, whereas at 50% cholesterol the membrane is entirely in the ordered phase. When increasing amounts of P-SM are added to the system, the required fraction of cholesterol to induce a complete l o phase drops from 50% (at 30% P-SM) to 35% (at 60% P-SM). The fact that the area of complete l o (phase) is largely above that of l d l o demonstrates that the main determinant of the ordered phase is cholesterol. Thus, upward movement in the diagram (which corresponds to increasing the cholesterol content) brings membranes closer to the area of entire ordered phase. It must be emphasized that there is no sudden change in the detergent resistance of membranes when the line between the areas in the phase diagram is passed. Schroeder et al 103 tested detergent (Triton X-100) resistance in membranes containing increasing amounts of SM (from 0% to 60%)

10 March 2006 HEPATOCANALICULAR TRANSPORT DEFECTS 917 Figure 3. Phase diagram for the (co-)existence of the liquid-disordered (l d ), the liquid-ordered (l o ), and solid-ordered (s o ) phases in membranes with various relative compositions of POPC, P-SM, and cholesterol. Abbreviations: POPC, phosphatidylcholine (containing 1 palimitic and 1 oleic fatty acyl chain); P-SM, sphingomyelin with a palmitic acyl chain. The solid square represents the composition of the basolateral membrane and the solid circle the composition of the canalicular membrane as purified and analyzed by Nibbering et al. 110 The arrow indicates the direction in which the canalicular membrane composition approximately changes if corrected for contaminating other membranes (mainly basolateral membranes and ER membranes). The upper (faint) part of the diagram represents relative cholesterol contents ( 66%) at which cholesterol is not soluble in the membrane. The figure was modified from Simons and Vaz 107 and de Almeida et al. 109 with a fixed amount of cholesterol (33%). With increasing amounts of SM detergent resistance linearly increased from 50% to 95%. According to the phase diagram the membranes are only in full l o configuration at the highest SM content (60%). Hence, already in the combined l d l o configuration there is a certain detergent resistance which becomes (nearly) complete when the membrane is in the l o configuration. Extrapolation of these data to the canalicular membrane is quite speculative, but provides important insights. First, POPC and P-SM are quantitatively important constituents of the canalicular membrane, 105,110,111 which means that in terms of bulk lipid, this phase diagram can be applied to this membrane. Evidently, the presence of proteins influences the behavior of these phases, but has not been taken into account here. If we plot into this diagram the composition of hepatic basolateral (solid square) and canalicular (solid circle) membranes isolated from mice, 110 it is clear that the canalicular membrane is much more near the border of an entire liquid ordered phase. Importantly, canalicular membrane preparations are not at all pure and main contaminants are basolateral membranes and membranes from the endoplasmic reticulum. The composition of basolateral membranes is given in the diagram (solid square) and contains less SM and less cholesterol than canalicular membranes (solid circle); ER membranes contain little if any cholesterol. Hence, the real canalicular membrane composition (as opposed to contaminated canalicular membranes as they are isolated) is shifted more to the right (higher SM content) and more upward (higher cholesterol content) as indicated by the arrow and, thus, closer to the area of entire l o phase. This is even more the case if one considers that the main determinant of detergent resistance is the outer leaflet, because this is the leaflet exposed to high bile salt concentrations. It is well known that sphingomyelin is not equally distributed over the 2 leaflets, but almost exclusively present in the outer leaflet. 112 As a consequence of the high affinity of cholesterol for SM, 109 cholesterol is enriched in the outer leaflet as well. This means that the outer leaflet composition of the canalicular membrane may well be in the liquid-ordered phase area of the diagram and thus be resistant toward detergent. Analysis of detergent resistance has mainly been performed with Triton X-100 and some other detergents.

11 918 OUDE ELFERINK ET AL GASTROENTEROLOGY Vol. 130, No. 3 Unfortunately, elaborate studies of this kind have not been performed with bile salts. Some studies do, however, suggest that a similar behavior does occur with bile salts. Hofmann et al 113 studied the diffusion of unconjugated CDCA into liposomes of different lipid composition and observed that addition of cholesterol to PC liposomes (1:1) caused a 4-fold reduction in the diffusion rate compared to pure PC. They also studied induction of liposome leakage by CDCA and found that this was 2-fold reduced by addition of equal amounts of cholesterol. Pure SM liposomes were in fact more sensitive to CDCA than pure PC liposomes. However, combination of the three lipids (SM/chol/PC 1:3:3) reduced the sensitivity by a factor of 4.8 (compared to pure PC) or even a factor of 35 (compared to pure SM). 113 A similar behavior with respect to the induction of leakage in liposomes, composed of these 3 components, was observed by Moschetta et al 114 using taurocholate as the detergent. These studies suggest a similar resistance of membranes containing SM and cholesterol toward bile salts as observed with Triton X-100. How Does Canalicular Cholesterol Translocation Fit in? Biliary cholesterol excretion in mice with a disrupted Abcg5 and Abcg8 gene is reduced by about 80% 28,115 ; similar observations have been made in mice with a disruption of only the Abcg8 gene 27,116 or only the Abcg5 gene (Plösch T, et al, in preparation), indicating that an obligate heterodimer of Abcg5 and Abcg8 forms a functional ABC transporter for cholesterol excretion into bile. 25 The discovery that yet another floppase is responsible for the biliary excretion of cholesterol has simplified matters on the 1 hand; on the other hand it complicated them. Although actual translocation of cholesterol by the transporter has not been demonstrated, it may be assumed on the basis of sequence homology that the Abcg5/g8 protein complex represents a transporter. Hence, the minimum hypothesis must be that Abcg5/g8 translocates cholesterol from the inner to the outer leaflet of the membrane and represents another floppase (Figure 2). It would, however, be one step further to assume that in the absence of these transporters no cholesterol is present in the outer leaflet of the canalicular membrane. First, it is known that spontaneous flip-flop of cholesterol across the membrane is much faster than that of phospholipids 117 and this was in fact for a long time an argument to assume that no transporter is required for biliary cholesterol excretion. Second, the crucial role of cholesterol in protection of the canalicular membrane against bile salts (as described) suggests that absence from the outer leaflet is impossible. If cholesterol were absent from the canalicular membrane outer leaflet, it would become highly vulnerable to bile salts. As a consequence, patients with sitosterolemia as well as Abcg5/g8 knockout animals should suffer from severe liver damage, similar to (or in fact stronger than) that observed in the Abcb4 / mouse and in PFIC3 patients. This is, however, not the case. In fact these animals and patients have no signs of liver damage at all. 27,28,118 The conclusion therefore must be that cholesterol is present in the canalicular membrane outer leaflet even when the Abcg5/g8 transporter is absent. Indeed, biliary cholesterol excretion is not completely absent in Abcg5/g8 knockout mice, but reduced to about 20% of wild-type levels, 27,28,115 suggesting that in the absence of the transporter still a significant amount of cholesterol can be extracted from the membrane. This is in stark contrast to the Abcb4 / mouse that completely lacks phospholipids in bile even when highly hydrophobic bile salts are infused. 36 In the case of PC, translocation is required and in the absence of Mdr2 there will be little if any PC in the outer leaflet. Nevertheless, the Abcg5/g8 transporter is apparently necessary for the bulk excretion of cholesterol; in its absence cholesterol secretion is impaired. Two lines of reasoning should be considered here. First, the transporter might be important to supply cholesterol to the outer leaflet only under conditions of increased flux, that is, during high bile salt excretion rates when cholesterol excretion should be high as well (floppase function). In line with this argument, it has been observed that increased bile salt flux, either by bile salt infusion 27 or bile salt feeding, 115 does not substantially increase the residual cholesterol excretion in the deficient mice (Figure 4). Second, it may be that simple extraction of cholesterol from the canalicular membrane by bile salts is not possible. In the context of the described lipid composition of the canalicular membrane, it may be expected that cholesterol molecules are buried in the sphingomyelin layer. 107 This increases the activation energy required for extraction so much that it cannot be accomplished by bile salts at body temperature. This possibility was already raised by Small, 119 who suggested that ABCG5/G8 might represent a liftase. The floppase and liftase models are certainly not mutually exclusive. Thus, in addition to translocating cholesterol from the inner to the outer leaflet of the membrane bilayer, the transporter may expose the cholesterol molecule sufficiently out of the bilayer to allow transfer to acceptor bile salt micelles.