“The many, often fantastic, theories advanced to explain jaundice without obstruction have long stood as clear evidence of the need of precise information regarding the formation and excretion of the bile ….”

A. R. Rich, Bulletin Johns Hopkins Hosp 1930;47:338

An American Association for the Study of Liver Diseases (AASLD)-sponsored Pediatric Single-Topic Conference focused on the category of hepatobiliary disease collectively known as syndromes of intrahepatic cholestasis (September 2004). A single-topic conference that focused on this topic was unique and timely because recent advances in molecular genetics have clearly pointed out new directions for investigation and clinical understanding. These disorders, which are individually rare, are common collectively; they are chronic (by definition) and many cases progress to end-stage disease, requiring liver transplantation. More effective treatment options are needed. Prevention and treatment strategies targeted to this age group will be especially cost-effective. It was the goal of the single-topic conference to help to promote further investigation to identify the mechanisms responsible for this subset of diseases that begin during childhood and lead to ongoing liver dysfunction in children and adults. The ultimate objective was to foster research and collaborative ventures in this important area. The conference brought together leading experts from around the world for presentations that highlighted the newest investigative and therapeutic approaches; the discussions drew needed attention to areas requiring further research. The purpose of this report is to briefly summarize this important single-topic conference.

Intrahepatic Cholestasis: From the Bedside to the Bench

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Proposed Subtypes of Genetic Intrahepatic Cholestasis

To place the topic in perspective, William F. Balistreri (Cincinnati Children's Hospital Medical Center) emphasized that in the early 1970s, the differential diagnosis of the neonate with conjugated hyperbilirubinemia (cholestasis) was limited. Biliary atresia accounted for approximately 25% of the cases; a small percentage of cases were considered to be caused by viral infections or were the result of a handful of recognizable metabolic or inherited diseases (i.e.,galactosemia, tyrosinemia, cystic fibrosis).1 The vast majority were designated as idiopathic neonatal hepatitis, clearly a default diagnosis, because the underlying pathophysiology was unknown. What was clear was that although many cases were apparently sporadic, others recurred in families. This led to the speculation that cases of familial neonatal hepatitis represented presumed inborn errors or genetic defects in a fundamental process involved in hepatic metabolic or excretory function. It followed that elucidation of their nature would allow a more clear understanding of liver physiology and also to improved therapy. Indeed, over the past 30 years there has been significant progress in dissecting the components of the idiopathic neonatal hepatitis spectrum. Specifically, substantial progress has been made in subcategorizing the range of hepatobiliary diseases that manifest as intrahepatic cholestasis in infants, children, and even adults.2 A major category is that of genetic intrahepatic cholestasis, a heterogeneous subset of diseases that represents specific disorders of abnormal cell fate, canalicular transport (bile acid or phospholipid), or bile acid synthesis, each with different prognostic implications. There are multiple forms with varying clinical features and with a high degree of variability in presentation and prognosis. Certain progressive, familial forms, as in the group known as progressive familial intrahepatic cholestasis (PFIC), are fatal; however, in patients with syndromic paucity of the ducts (Alagille syndrome), the prognosis is more favorable.2 The current system of nomenclature for the various syndromes in which intrahepatic cholestasis is present is imperfect. A proposed classification scheme is shown in Table 1. These syndromes are of more than theoretical interest; detailed study of affected patients has enhanced our understanding of hepatic excretory function and bile acid metabolism. The pathogenetic basis for these diseases has been defined only partially, and the techniques of molecular genetics have been applied only recently to these disorders. Precise terminology of the intrahepatic cholestatic disorders, based on the documented genetic defect, not only will provide clues to the pathophysiology but will also help to establish registries, promote development of new treatments, and allow the institution of valid clinical therapeutic trials.

Table 1. Proposed Subtypes of Intrahepatic Cholestasis
  • NOTE. FIC1 deficiency, BSEP deficiency, and some of the disorders of bile acid biosynthesis are characterized clinically by low levels of serum GGT despite the presence of cholestasis. In all other disorders listed, the serum GGT level is elevated.

  • Abbreviations: ADPLD, autosomal-dominant polycystic liver disease (cysts in liver only); ARPKD, autosomal-recessive polycystic kidney disease (cysts in liver and kidney); BAAT, bile acid transporter; BRIC, benign recurrent intrahepatic cholestasis; PFIC, progressive familial intrahepatic cholestasis.

  • *

    See Table 2.

A. Disorders of membrane transport and secretion
 1. Disorders of canalicular secretion
  a. Bile acid transport—BSEP deficiency
   i. Persistent, progressive (PFIC Type 2)
   ii. Recurrent, benign (BRIC Type 2)
  b. Phospholipid transport—MDR3 deficiency (PFIC Type 3)
  c. Ion transport—cystic fibrosis (CFTR)
 2. Complex or multiorgan disorders
  a. FIC1 deficiency
   i. Persistent, progressive (PFIC type 1, Byler's disease)
   ii. Recurrent, benign (BRIC type 1)
  b. Neonatal sclerosing cholangitis (CLDN1)*
  c. Arthrogryposis-renal dysfunction-cholestasis syndrome (VPS33B)*
B. Disorders of bile acid biosynthesis and conjugation
 1. 3-oxo-4-steroid 5β-reductase deficiency
 2. 3 β-hydroxy-5-C27-steroid dehydrogenase/isomerase deficiency
 3. Oxysterol 7 α-hydroxylase deficiency
 4. BAAT deficiency (familial hypercholanemia)*
C. Disorders of embryogenesis
 1. Alagille syndrome (Jagged 1 defect, syndromic bile duct paucity)
 2. Ductal plate malformation (ARPKD, ADPLD, Caroli's disease)
D. Unclassified (idiopathic “neonatal hepatitis”)—mechanism unknown
Table 2. Molecular Defects in Intrahepatic Cholestasis
GeneProteinFunction, SubstrateDisorderReference
  1. Abbreviations: ADPLD, autosomal dominant polycystic liver disease; ARC, arthrogryposis-renal dysfunction-cholestasis syndrome*; ARPKD, autosomal recessive polycystic kidney disease; BAS, bile acid synthetic defect; BRIC, benign recurrent intrahepatic cholestasis; CFTR, cystic fibrosis transmembrane conductance regulator; FHC, familial hypercholanemia; GFC, Greenland familial cholestasis; ICP, intrahepatic cholestasis of pregnancy; NAICC, North American Indian childhood cirrhosis; NSC, neonatal sclerosing cholangitis with ichthyosis, leukocyte vacuoles, and alopecia; PFIC, progressive familial intrahepatic cholestasis.*

  2. *Low GGT (PFIC types 1 & 2, BRIC types 1 & 2, ARC).

ATP8B1FIC1P-type ATPase; aminophospholipid translocase that flips phosphatidylserine and phosphatidylethanolamine from the outer to the inner layer of the canalicular membranePFIC1 (Byler's disease), BRIC1, GFC16, 17
ABCB11BSEPCanalicular protein with ATP binding cassette (ABC family of proteins); works as a pump transporting bile acids through the canalicular domainPFIC 2, BRIC 218–20
ABCB4MDR3Canalicular protein with ATP binding cassette (ABC family of proteins); works as a phospholipid flippase in canalicular membranePFIC3, ICP, Cholelithiasis19, 21–23
AKR1D15β-reductase3-oxoΔ-4-steroid 5β-reductase gene; regulates bile acid synthesisBAS: Neonatal cholestasis with giant cell hepatitis24
HSD3B7C27-3β-HSD3β-hydroxy-5-C27-steroid oxido-reductase (C27-3β-HSD) gene; regulates bile acid synthesisBAS: Chronic intrahepatic cholestasis3, 25
CYP7BICYP7BIOxysterol 7α-hydroxylase; regulates the acidic pathway of bile acid synthesisBAS: Neonatal cholestasis with giant cell hepatitis3, 26
JAG1JAG1Transmembrane, cell-surface proteins that interact with Notch receptors to regulate cell fate during embryogenesisAlagille syndrome27, 28
TJP2Tight junction proteinBelongs to the family of membrane-associated guanylate kinase homologs that are involved in the organization of epithelial and endothelial intercellular junction; regulates paracellular permeabilityFHC29, 30
BAATBAATEnzyme that transfers the bile acid moiety from the acyl-CoA thioester to either glycine or taurineFHC30
EPHX1Epoxide hydrolaseMicrosomal epoxide hydrolase regulates the activation and detoxification of exogenous chemicalsFHC31
ABCC2MRP2Canalicular protein with ATP binding cassette (ABC family of proteins); regulates canalicular transport of GSH conjugates and arsenicDubin-Johnson syndrome32
ATP7BATP7BP-type ATPase; functions as copper export pumpWilson disease33
CLDN1Claudin 1Tight junction proteinNSC34
CIRH1ACirhinCell signaling?NAICC35
CFTRCFTRChloride channel with ATP binding cassette (ABC family of proteins); regulates chloride transportCystic Fibrosis36
PKHD1FibrocystinProtein involved in ciliary function and tubulogenesisARPKD37–38
PRKCSHHepatocystinAssembles with glucosidase II alpha subunit in endoplasmic reticulumADPLD39
VPS33BVascular Protein Sorting 33Regulates fusion of proteins to cellular membraneARC40

Liver Pathology in Intrahepatic Cholestasis

Kevin E. Bove (Cincinnati Children's Hospital Medical Center) emphasized that these genetic defects result in lobular cholestasis, variable giant cell transformation (GCT) of liver cells, isolated hepatocyte necrosis, and a highly variable rate of progression to fibrosis. The degree of hepatocellular injury is least in presumed cases of FIC1 deficiency, intermediate in bile salt export pump (BSEP) deficiency, and most destructive in MDR3 deficiency. In routine practice, the diagnosis is tentative and dependent on clinical parameters (i.e., serum γ-glutamyltransferase [GGT] and bile acid level, phenotype), combined with liver biopsy findings using light and electron microscopy.2 He discussed the predominant liver morphological features of the various forms. In FIC1 deficiency, small tidy hepatocytes with minimal ballooning and bland canalicular cholestasis are present; GCT is uncommon; there is little or no cholangiolopathy (periportal ductular metaplasia) with slow progression of portal fibrosis; and an unusual coarse quality of bile in canaliculi is visualized by electron microscopy. In BSEP deficiency, prominent canalicular cholestasis is often zonal (zone 3 > zone 1); prominent clustered balloon cholestasis of hepatocytes is present; GCT is common and persists beyond infancy; perivenular, pericellular, and periportal fibrosis with progression to cirrhosis is present; mild cholangiolopathy is present; normal interlobular bile ducts are present; and delicate wispy nonspecific quality of bile in canaliculi by electron microscopy is evident. In MDR3 deficiency, there is cholestatic hepatitis with GCT in infants and young children, cholangiolopathy, and progressive injury of the interlobular bile ducts in the early stages of disease. In bile acid synthetic defects, Dr. Bove has noted generalized hepatocyte injury with GCT and periportal inflammatory fibrogenic cholangiolitis; the injury and progressive portal fibrosis with fusion or dysmitotic division of immature hepatocytes is attributed to atypical or toxic bile acids present in the liver in conjunction with reduced concentrations of normal bile acids.3 In Alagille syndrome, he has observed that the smallest ductules either fail to form or rapidly regress, with interlobular bile ducts subject to a smoldering cholangiopathy that results in focal proliferation and progressive destruction at a variable rate.

Molecular Basis of Canalicular Transport

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

The next session focused on potential mechanisms responsible for the clinical and histological features described above. The predominant mechanism in many forms of intrahepatic cholestasis is altered canalicular transport.

Composition and Function of the Bile Canaliculus

The bile canaliculus is surrounded by the apical plasma membrane domains of two or three hepatocytes (Fig. 1).4, 5 The composition of the canalicular membrane is distinct from the basolateral membrane and is enriched in cholesterol and sphingomyelin. Canalicular membrane proteins are targeted to this domain via both direct and indirect pathways. The canalicular membrane is enriched in ABC transporters that function as export pumps for bile acids and a variety of organic solutes. James Boyer (Yale University School of Medicine) pointed out that this membrane domain is metabolically active because it contains a number of active ATP-dependent solute transport proteins as well as GPI-anchored proteins like dipeptidyl peptidase IV, GGT, aminopeptidase N, 5′-nucleotidase, and the protein HA-4. Ion and water channels, ion exchangers, skeletal proteins (villin), vesicle fusion proteins (e.g., SNARE, SNAP, Syntaxin, Rabs) and tight-junction proteins are also present. Most of the solute transport-proteins belong to the superfamily of ABC transporters. The BSEP (ABCB11) is one; BSEP mediates the transport of conjugated bile acids (cholic and chenodeoxycholic acid). Like all other plasma membrane proteins, BSEP is synthesized in the endoplasmic reticulum and after glycosylation in the trans-Golgi network travels through the subapical compartment to the canalicular membrane. There is evidence for an intracellular pool of BSEP protein from which BSEP may be recruited on demand. For retraction and internalization of BSEP, interaction with a protein called HAX-1 seems to be important.4 Other apical ABC transporters include MRP2 (ABCC2), which transports glucuronide and glutathione conjugates; MDR3 (ABCB4), a flippase of phosphatidylcholine; MDR1 (ABCB1), a transporter of chemotherapeutic agents; ABCG5G8, a heterodimeric protein that transports sterols; and BCRP breast cancer resistance protein (ABCG2), a transporter of chemotherapeutic agents, carcinogens, and natural products.5–8 Mutations in several of these ABC transporters (MDR3, BSEP, and MRP2) result in clinical disease.

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Figure 1. The bile canalicular and the major hepatobiliary transport systems. The basolateral membrane of the hepatocyte contains the solute carrier systems (organic anion transport proteins [OATPs]), whereas the ATP-dependent ABC transporters are mainly located in the canalicular membrane. During cholestasis, multidrug resistance proteins MRP3 and MRP4, two organic anion transporting systems, become expressed at the basolateral membrane. MRP3 mainly transports conjugates such as bilirubin glucuronide; MRP4 transports bile acids together with glutathione. Transport proteins, water and electrolyte channels, and exchangers at the bile duct epithelium indicate that bile composition is modified at this level.

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The function of transporters in the basolateral membrane is quite different from those at the apical membrane. Uptake from the portal blood into the liver does not require active transport; electroneutrality is sufficient. Some of these transporters are exchangers that mediate organic anion uptake in exchange for bicarbonate or glutathione. These proteins belong to the superfamily of solute carriers (organic anion transport proteins [OATPs]) named OATP-1 (Slc21a1), OATP-2 (Slc21a5), OATP-4 (Slc21a6), and OATP-8 (Slc21a8). They have broad and partly overlapping substrate specificity. NTCP (Slc10a1) is an exception; it transports only bile acids and is a sodium–bile acid cotransporting protein.7 Solute carriers reach the basolateral membrane via a direct sorting route from the trans-Golgi. Their expression is dependent on transcriptional and post-transcriptional regulatory mechanisms (Fig. 2). MRP3 (Abcc3) and MRP4 (Abcc4) in the basolateral membrane act as efflux pumps when canalicular secretion is impaired. MRP4 is a cotransporter of bile acids and glutathione, whereas MRP3 mainly transports conjugates such as bilirubin monoglucuronide and diglucuronide. These transporters have a low expression in normal liver but are upregulated in cholestatic conditions.9, 10 Much remains to be learned about the mechanisms of canalicular membrane formation in development and its maintenance in health and disease.

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Figure 2. Post-transcriptional regulation of canalicular transporters. Apical transport proteins cycle between the apical membrane and a subapical compartment (SAC). On hypoosmotic conditions and/or stimulation by cyclic-AMP, protein kinase C, phosphatidylinositol-3-kinase, or MAP-kinase, transport proteins are inserted into the apical membrane. On hyperosmotic conditions, LPS administration, or bile duct ligation, they are retrieved into a SAC. The regular half-life of a transport protein is governed by proteosomal degradation after ubiquination. Drugs may influence the activity of transporters by direct interference with their activity. They can do this from the cytoplasmic side (cis-inhibition) or from the canalicular lumenal side (trans-inhibition). Modified from Trauner and Boyer.5

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Ontogeny and Regulation of Bile Acid Transporters

Frederick J. Suchy (Mount Sinai Medical Center, New York) discussed the ontogeny and regulation of these bile acid transporters. At birth, hepatic excretory function is immature, the enterohepatic circulation is inefficient, and bile flow is reduced; immediately after birth, serum bile acid levels are elevated.11 Ntcp and Bsep messenger RNA (mRNA) expression is not fully developed until 1 week after birth (rats). Ntcp protein expression closely follows this mRNA expression. Bsep protein expression is somewhat delayed and is not fully developed until 4 weeks after birth. The same holds true for Mrp2 mRNA and protein.12 The genes encoding these proteins are under transcriptional control of the nuclear hormone receptors RXR, FXR, PXR, CAR, RAR, LXR, and LRH. These are transcription factors that translocate from the cytoplasm to the nucleus on binding a relevant ligand. Bile acids bind to FXR, drugs to CAR and PXR, retinoic acid to RARα,β,γ, and oxysterols to LXRα,β. LRH-1 is an orphan receptor with no known ligand. 9-Cis-retinoic acid binds to RXRα,β,γ. In the nucleus, they interact as heterodimers with palindromic response elements in the promotor regions of target genes. RXRα,β,γ are obligate partners.13 What has been said about the ontogeny of Ntcp, Bsep, and Mrp2 is also true for the nuclear hormone receptors; their expression is significantly less than adult values until 1 week after birth.14 Because these receptors play an important role in protecting the liver against substrate overload, it holds that in the first week of life, the neonate is particularly susceptible to cholestasis and liver damage.2 The exact mechanisms governing the ontogeny of transport proteins and metabolizing enzymes is not known. A complex interaction of nuclear hormone receptors and other transcription factors is likely to play a role. It is clear that nuclear hormone receptors do not act alone but are part of a complex of coactivating proteins. Coactivator-associated arginine methyltransferase (CARM1) acts as a coactivator of FXR, activating BSEP. FXR:RXR and CARM1 are needed for full expression of BSEP mRNA in Hep G2 cells.15

Molecular Defects as the Basis of Cholestatic Syndromes

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Given the complexity of the structure and function of the bile canaliculus and hepatic excretory systems, it has long been postulated that specific inborn molecular defects could lead to cholestasis. A large number of genes mutated in forms of intrahepatic cholestasis have been identified (Table 2)16–40; however, many as-yet unidentified cholestasis genes likely exist.

Laura Bull (University of California San Francisco, San Francisco General Hospital) described genetic approaches to decipher disorders such as intrahepatic cholestasis. These approaches complement functional studies of hereditary diseases. Bull emphasized that a key feature of genetic mapping studies is that they permit identification of the genetic etiology of a disease, even if the identity of the disease gene could not have been predicted. Thus, such studies can facilitate our understanding of disease etiology. For example, it is unlikely that FIC1/ATP8B1 would have been easily identified as a cholestasis gene using a functional, rather than whole-genome screening, approach. Bull has carried out genetic mapping studies to sift through the approximately 3.2 gigabasepairs of DNA in a single copy of the genome to identify mutations that result in disease.41

Molecular Defects in Disorders of Canalicular Transport

FIC1 Deficiency.

Roderick Houwen (Wilhelmina Children's Hospital, Utrecht, The Netherlands) discussed the clinical relevance of FIC1 (ATP8B1), the gene involved in various forms of intrahepatic cholestasis associated with FIC1 deficiency (PFIC type 1, Byler's disease, Greenland familial cholestasis, and benign recurrent intrahepatic cholestasis [BRIC] type 1).42, 43 Both PFIC type 1 and BRIC type 1 are autosomal recessive diseases. Nonsense, frameshift, and deletional mutations cause PFIC type 1, whereas missense and splice site mutations cause BRIC type 1. However, identical mutations can cause both PFIC type 1 and BRIC type 1. Houwen emphasized that homozygosity for the common I661T mutation can also occur in healthy persons. This indicates that modifier genes and environmental influences play a role in the expression of PFIC type 1 and BRIC type 1. The pathophysiology of these diseases is not well understood. FIC1 encodes an aminophospholipid translocase that has been proposed to flip phosphatidylserine and phosphatidylethanolamine from the outer to the inner layer of the plasma membrane. How a defect of this protein leads to cholestasis is unknown, and studies carried out on FIC1 knockout mice have failed to elucidate the mechanism.44 FIC1 protein expression in the intestine exceeds that in the liver, and therefore defects in both intestine and liver may be involved in the pathogenesis of these diseases. Increased intestinal absorption of bile acids, decreased canalicular secretion of bile acids, or both may be implicated. Partial biliary diversion, in which the gallbladder is externalized via a stoma using a loop of small intestine, is of clinical benefit in approximately half of the patients with PFIC type 1.45 The bile acid pool is reduced via diversion of bile, but how this leads to amelioration of cholestasis is unknown.

BSEP Deficiency.

Richard Thompson (Guy's, King's & St Thomas' School of Medicine, London) discussed BSEP (ABCB11), the major canalicular bile acid transporter. Mutations of the BSEP gene lead to deficiency of canalicular BSEP expression and variable degrees of intrahepatic cholestasis (also known as PFIC type 2 and BRIC type 2). As in FIC1 deficiency (PFIC type 1 and BRIC type 1), serum GGT activity in these patients is not elevated despite the cholestasis. A great number of mutations have been described in patients with PFIC type 2.18, 46 As in PFIC type 1, severe mutations lead to a phenotype of unremitting cholestasis from birth onward. Missense mutations have been shown to cause a milder disease not unlike BRIC; follow-up studies of this group of patients has not been long enough to know if this disease runs the same benign course as BRIC type 1. Thompson also reported eight patients with PFIC type 2 in whom malignancy developed: five with hepatocellular carcinoma, one with hepatoblastoma, and two with cholangiocarcinoma. The pathogenesis of PFIC type 2 seems clear; deficiency of the major canalicular bile salt transporter leads to severe cholestasis. Therefore, it was surprising that Bsep knockout mice are viable and in fact have a very mild phenotype.47, 48 Studies on these mice made it clear that mice have compensatory pathways to eliminate or detoxify bile acids, or both; for example, tetrahydroxy bile acids are produced that can be eliminated via alternative routes. Humans have the ability to upregulate the expression of basolateral MRP4 as a hepatic escape mechanism. Treatment of PFIC type 2 is mainly supportive, followed by liver transplantation in many cases. The role of partial biliary diversion has to be established; some patients respond well to this therapy, particularly those with the D482G or E297G mutation.20, 42

MDR3 Deficiency.

Ronald Oude Elferink (Academic Medical Center, Amsterdam, The Netherlands) was involved in the discovery of MDR3/Mdr2 (ABCB4/Abcb4) as a phospholipid translocase that flips phosphatidylcholine from the inner to the outer layer of the canalicular membrane.23, 49 Mutations of the MDR3 gene lead to the formation of phosphatidylcholine-poor bile with a normal bile acid concentration. The high content of bile acids in the absence of phospholipid may make this bile toxic to hepatocytes and to the bile duct epithelium. Patients and mice with a defective canalicular MDR3/Mdr2 expression show severe hepatocyte and bile duct damage, periductular inflammation, portal and periportal fibrosis, and eventually (in mice) bile duct carcinoma. The phenotypic variation of this disease is remarkable and includes intrahepatic gallstone formation, biliary fibrosis, and intrahepatic cholestasis of pregnancy. In humans, the cholestatic syndrome that is associated with MDR3 mutations is called MDR3 deficiency or PFIC type 3. In contrast to PFIC type 1 and PFIC type 2, PFIC type 3 is characterized by increased serum GGT levels. A number of PFIC type 3 patients, presumably with partial residual MDR3 function, respond well to ursodeoxycholate therapy.50

Developmental Abnormalities of the Biliary System

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Certain forms of intrahepatic cholestasis have been ascribed to altered embryogenesis; the mechanisms and features of these developmental abnormalities of the biliary system were discussed.

Molecular Defects in Patients With the Alagille Syndrome

The most common cause of chronic cholestasis during childhood associated with specific phenotypic features is the Alagille syndrome. There is wide variability in the extent of phenotypic expression; affected patients may present with any combination of five major features: (1) chronic cholestasis resulting from a paucity of interlobular bile ducts, (2) cardiovascular malformations, such as peripheral pulmonic stenosis or more severe lesions as in tetralogy of Fallot, (3) vertebral arch defects, (4) posterior embryotoxon and other ocular abnormalities, and (5) unique facial features.51 In addition, the kidney, pancreas, and cerebrovascular system may be involved. The disease is often manifest as neonatal cholestasis, with pruritus and xanthomas becoming prominent symptoms in later phases of disease; hepatic and cardiac manifestations are responsible for most morbidity and mortality. The genetic defect in patients with the Alagille syndrome has been shown to reside in mutations of the Jagged-1 gene.27, 28

Nancy Spinner (The Children's Hospital of Philadelphia) reviewed how mutations in the Jagged-1 gene correlate with the variable manifestations in patients with the Alagille syndrome. Since the initial identification of mutations capable of disrupting the product of Jagged-1 in affected patients,27, 28 several studies have explored the extent to which mutations segregate with disease phenotypes. Jagged-1 belongs to the Jagged family of genes that encode cell surface proteins that interact with Notch receptors, generating signals to regulate cell fate during embryogenesis. To date, genetic screening techniques have detected Jagged-1 mutations in 60% to 70% of individuals with hepatic, cardiovascular, and abnormalities in other systems.52, 53 However, higher detection rates are now possible. These mutations are spread out across the coding region, and include total gene deletions, protein truncating mutations (caused by nonsense, insertion, and deletion mutations), splicing mutations, and missense mutations. Mutations are de novo in more than 50% of the cases; although most mutations occur in the extracellular domain of the JAG1 protein, there is no predominant mutational “hot spot,” making it difficult to routinely use genetic testing in the evaluation of patients with the Alagille syndrome. At the molecular level, haploinsufficiency (decreased gene dosage) seems to be the mechanism of clinically relevant mutations. Notably, missense mutations, which account for 10% to 15% of all mutations, lead to intracellular trafficking defects, with trapping of the mutant protein in the perinuclear region, thus preventing the proper membrane anchoring at the cellular surface. Despite the greater understanding of the molecular consequences of mutations, the search for phenotype-causing mutations has been elusive, with no correlation between mutations and the phenotypic expression in patients with the Alagille syndrome. One exception is the high association of the JAG1-G274D mutation with a cardiac specific phenotype, supporting the concept that specific Jagged-1 mutations may have greater penetrance in the developing heart.54, 55 Specific Jagged-1 mutations that segregate exclusively with hepatic abnormalities have not yet been reported. This notwithstanding, an important role for the JAG–Notch pathway in the hepatobiliary system is supported by findings of abnormal morphogenesis of the intrahepatic bile ducts in experimental models. For example, mice with dual heterozygosity for the Jagged-1 gene and of its receptor Notch-2 exhibit a phenotype akin to the Alagille syndrome.56 Interestingly, inhibition of gene expression leading to interruption in the JAG-mediated Notch signaling was recently shown to also result in abnormal development of the intrahepatic biliary system.57

Molecular Control of Biliary Development and the Ductal Plate Malformation

Frederick Lemaigre (Universite Catholique de Louvain, Brussels, Belgium) and Ai-Xuan Holterman (University of Illinois at Chicago) reviewed recent data identifying additional molecular networks that play key regulatory roles in the morphogenesis of the biliary system.

Abnormalities in morphogenesis restricted to intrahepatic bile ducts (ductal plate) have been demonstrated in mice with disrupted JAG-Notch signaling, as discussed above.56, 57 In contrast, developmental abnormalities restricted to the extrahepatic biliary system have been found in Hes1-deficient mice.58Hes1 encodes the basic helix-loop-helix protein Hes1, which is expressed in the extrahepatic biliary epithelium throughout development. Interestingly, Hes1 expression is controlled by the Notch pathway; however, in contrast to the predominantly intrahepatic phenotype when the JAG-Notch pathway is disrupted, in vivo loss of Hes1 results in gallbladder agenesis and severe hypoplasia of extrahepatic bile ducts.

Despite the distinct endodermal origin of the intrahepatic and extrahepatic biliary systems, the molecular forces driving tissue embryogenesis are shared, at least in part, by common pathways, as demonstrated by the phenotypic analysis of mice carrying the inactivation of genes encoding members of two groups of transcription factors. The first is the hepatocyte nuclear factors (HNF)-6 and HNF1β. HNF6, a member of the onecut family of homeoproteins, is expressed in intrahepatic biliary cells and gallbladder primordium. When the HNF6 gene was inactivated in mice, development of the ductal plate was impaired, biliary cysts developed, the gallbladder did not form, and extrahepatic bile ducts were replaced by an enlarged tubular structure that connected the liver to the duodenum.59 HNF6 is known to control the expression of HNF1β. The functional relationship between these factors and relevance to biliary development were further established by the findings of paucity of small intrahepatic bile ducts, dysplasia of larger intrahepatic bile ducts, and abnormal gallbladder and cystic duct in mice lacking HNF1β.60 These findings led to the proposal that the HNF6 [RIGHTWARDS ARROW] HNF1β pathway is essential for biliary development. It is likely that the application of similar experimental strategies to other transcription factors that are functionally related and expressed in the developing biliary system, such as the factor Onecut-2, will further define how HNFs work in concert to promote biliary development.

The second group of transcription factors is the Forkhead Box (Fox) family, known to participate in a broad range of physiological processes such as proliferation, differentiation, transformation, metabolism, and development. In the hepatobiliary system, the factor Foxf1 is expressed in stellate cells and in mesenchymal cells surrounding the intrahepatic and extrahepatic bile ducts. The importance of this expression pattern to biliary development became apparent in gene targeting experiments. Although homozygous inactivation of the Foxf1 gene was embryonic lethal owing to impaired extraembryonic tissue maturation, some heterozygous littermates completed gestation and were later found to have normal intrahepatic bile ducts, but diminutive or absent gallbladder.61 Conversely, targeting of the related gene Foxm1b resulted in impaired development of intrahepatic bile ducts, possibly because of the inability of embryonic hepatoblasts to differentiate toward biliary epithelial cell lineage.62

Collectively, the ability to inactivate genes in vivo and at different phases of embryonic development has facilitated the initial dissection of the molecular circuits controlling morphogenesis of the biliary system (Table 3). The implication of an increased understanding of the molecular basis of biliary development to clinical practice is highlighted by the direct relationship of the JAG-Notch circuit with the Alagille syndrome. In the future, analysis of these molecules in children with syndromes of intrahepatic and extrahepatic cholestasis will explore their potential role as cause or modifiers of disease pathogenesis.

Table 3. Molecular Circuits Controlling Morphogenesis of the Biliary System
  • Abbreviations: IHBD, intrahepatic bile ducts; EHBD, extrahepatic bile ducts.

  • *


Jagged-Notch circuitAbnormalUnaffectedUnaffected
HNF6Ductal plate malformationAbnormalAgenesis
 Intrahepatic biliary cysts  
HNF1βPaucity of small IHBD?*Abnormal epithelium
 Dysplasia of larger IHBD Dilated cystic duct
Foxf1Normal?*Small or absent
   No epithelial cells

Insight From Global Genomics and Phenotypic Expression

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Further understanding of the disorders of intrahepatic cholestasis will require insight from global genomics and phenotypic expression.

Genomics: Uncovering Clues of Biology and Phenotype

Jorge Bezerra (Cincinnati Children's Hospital Medical Center) discussed the use of molecular profiling through gene microarrays to study liver biology and pathobiology in specific clinical phenotypes. To date, more than 400 such studies have focused on subjects such as liver regeneration, development, carcinogenesis, inflammation, metabolism, and viral hepatitis. Experimental studies have also been carried out on the topic of cholestasis in both humans and animal models. Although this experimental approach has extraordinary potential in elucidating the genetic basis of disease as well as the transcriptional regulation of biological processes, it was emphasized that very sophisticated bioinformatic support is essential to analyze the very large amount of data typically acquired. Bezerra gave several examples of his own work that emphasizes the power of this approach; he and his colleagues initially applied high-density microarrays to determine the consequences of biliary obstruction on liver gene expression.63 Using the model of bile duct ligation in adult mice, he examined the level of expression of approximately 12,000 genes and generated a molecular footprint reflecting the activation of three biological processes that manifest in a sequential and time-restricted fashion. Initial changes were identified in genes involved in sterol metabolism. This was followed several days later by an increased expression of growth-promoting genes that corresponded to a point of peak cholangiocyte proliferation. By 14 to 21 days, when the liver displayed increased deposition of extracellular matrix, there was a unique increase in genes encoding structural proteins and proteases.

Bezerra and associates then studied gene expression profiling in infants with biliary atresia. The initial hypothesis tested was that there would be differences in gene regulation in the two clinical forms of biliary atresia: the embryonic form and the acquired form.63 Analysis of liver samples at the time of diagnosis successfully identified a highly distinctive gene expression profile. In the embryonic form, a coordinated expression of regulatory genes was observed, including those in chromatin integrity/function. Moreover, five imprinted genes were also overexpressed, implying a possible failure to downregulate embryonic gene programs. Functional analysis of the profile did not reveal notable differences between the clinical forms in the commitment of inflammatory cells or level of expression of genes determining laterality. Genetic profiling was also used to better define pathogenetic mechanisms involved in the acquired form of biliary atresia. When liver gene expression in children with biliary atresia was compared with that of patients with other forms of neonatal cholestasis, an increase in expression of genes involved in inflammation and immunity was defined.64 There was increased expression of interferon gamma and a profile consistent with a Th1-immune response. To directly test the hypothesis that a proinflammatory process and interferon gamma were involved in producing hepatobiliary injury in infants with biliary atresia, Bezerra and colleagues examined these pathways in a mouse model in which infection with rotavirus on the first day of life results in biliary atresia within 7 days.65 The infection triggered hepatobiliary inflammation by interferon gamma producing CD4-positive and CD8-positive lymphocytes. The inflammation was specific and limited to the liver, producing obstruction of the extrahepatic bile duct. In animals with genetic loss of interferon gamma production, jaundice still occurred but the tissue-specific targeting of lymphocytes was attenuated and the inflammatory and fibrosing obstruction of the bile ducts was prevented. Administration of recombinant interferon to these animals restored bile duct obstruction after rotavirus infection. These studies, directed by findings in patients with biliary atresia, provide support to the concept that the immune response driven by interferon gamma is an important pathogenetic mechanism producing bile duct destruction.66

Proteomics of Membrane Transporters

Barry Rosen (Wayne State University School of Medicine) gave an overview of membrane transporters with particular attention to his main area of research, arsenic transporters.67, 68 He first discussed P-type ATPases and the possible function of FIC1, the transporter that is defective in PFIC type 1. The function of this transporter is not known; Rosen discussed the possibility that FIC1 flips aminophospholipids from the outer to the inner leaflet of membranes. It is also possible that the FIC1 transports an ion that is coupled to aminophospholipid movement. The lack of membrane asymmetry that could be mediated by this transporter protein may disrupt other transporters leading to cholestasis. As a model, Rosen presented information of the pathways of arsenic uptake and efflux by the liver69, 70; his recent studies indicated a model of arsenic movement through hepatocytes that begins with uptake by channels, carriers, or both and ends with excretion into bile by ABC transporters.68 He has demonstrated that human AQP9, the major aquaglyceroporin, is a channel for arsenite.71 The transport of arsenite compounds by human AQP9 could be demonstrated in both yeast and xenopus oocytes.72 Two mammalian transporters of the ABC binding cassette family, MRP1 and MRP2, have been shown to pump glutathione conjugates of arsenite. Rosen and his colleagues have identified MRP homologs in yeast and other organisms that are responsible for arsenite detoxification.70 Other P-type ATPases are involved in transporting metals in bacteria, including a close homolog of ATP7B that is responsible for Wilson's disease.33

Genetic Polymorphisms Modulating Disease Phenotype

A number of polymorphisms in genes encoding immunoregulatory proteins, cytokines, and fibrogenic factors can modify the hepatic response to a variety of insults including alcohol, toxins, and viral hepatitis.73

David Brenner (Columbia University College of Physicians & Surgeons) discussed genetic polymorphisms modulating disease phenotype; he used hepatic fibrosis as a paradigm.74 Recent human epidemiologic evidence indicates possible polymorphisms in a number of candidate genes that influence the progression of liver fibrosis. These polymorphisms involve genes encoding immunoregulatory proteins, proinflammatory cytokines, and fibrogenic factors. Knockout mice have provided insight into factors that can modify hepatic fibrosis. Susceptibility to fibrosis is increased in interleukin-6, interleukin-10, inducible nitric oxide synthase, plasminogen, interferon gamma, telomerase, and adiponectin knockout mice. In contrast, susceptibility to fibrosis is decreased in TNFR1, transforming growth factor (TGF)-β1, Smad, leptin, OB-R, FasL, cathepsinB, angiotensin R1A, and angiotensin R1B null mice. As a specific example, TGFβ1 has been shown to activate hepatic stellate cells.74 TGFβ1 stimulates the production of extracellular matrix including collagens, glycoproteins, and proteoglycans; it also inhibits extracellular matrix degradation by inhibiting matrix metalloproteinases and stimulating protease inhibitors. In TFGβ1 knockout mice, the production of hepatic collagen α1 mRNA is markedly reduced on exposure to carbon tetrachloride. TGFβ1 signaling acts through Smad proteins. Similar to TGFβ1 knockout mice, Smad3 null mice demonstrate a lower production of hepatic collagen α1 and collagen α2 mRNA than controls after exposure to carbon tetrachloride.

Adiponectin, the most abundant tissue protein found in serum, mediates insulin sensitivity. Adiponectin treatment improves alcohol-induced dyslipidemia in mice as well as alcohol-induced hepatic steatosis and inflammation by decreasing the production of TGFβ1 and increasing hepatic fatty acid oxidation. Adiponectin knockout mice develop more extensive hepatic fibrosis than wild-type mice on exposure to carbon tetrachloride, have higher levels of TGFβ1, and demonstrate greater activation of hepatic stellate cells.75 The administration of adiponectin blocks hepatic stellate cell proliferation, migration, and fibrogenic signaling in wild-type mice treated with carbon tetrachloride. Adiponectin also prevents endotoxin-induced hepatic injury by inhibiting synthesis, the release of tumor necrosis factor α in an obese mouse model, or both.76

Cystic fibrosis-associated liver disease was cited as an example in which a disease-modifier gene may be operative. A recent correlation has been made between cystic fibrosis-associated liver disease and α1-antitrypsin genotypes. A multicenter study found that in 118 cystic fibrosis patients with CFTR mutations on both alleles, there was a threefold to sevenfold increased risk of developing severe liver disease if the patient carried the PIZ genotype (Knowles M, 2004, unpublished observation). A second modifier gene was also identified, a variant leading to increased production of TGFβ1, which potentially interacts with PIZ, resulting in an adverse impact on cystic fibrosis-associated liver disease. TGFβ1 mRNA is increased in the liver of patients with chronic hepatitis C virus infection in comparison with healthy controls, and the level of expression has been shown to correlate with expression of type 1 collagen mRNA.74

In searching for candidate genes that may be disease modifiers in intrahepatic cholestasis, there are several criteria that must be fulfilled. First, a gene product should play a role in disease pathogenesis. Mutations in the gene may be detected in familial forms of the disease. Knockout or overexpression in animal models should modulate disease development or activity. The gene should also lie in a chromosomal region associated with disease in a linkage study. Gene expression should be altered in tissue from patients with the disease. It is also desirable that the gene is found to be mutated in a phenotype-driven mouse study. Brenner also emphasized that case and control selection is critical. The disease phenotype should allow robust classification of cases and controls. Cases and controls should be matched and be derived from a similar genetic background or steps should be taken to adjust for population stratification. Confounding factors should be accounted for, either in matching cases and controls or should be included in the analysis of the gene effect. The stage of disease should also be taken into account in analyzing the effect of a susceptibility gene. For positive studies, the evidence should be strong enough to exclude chance. Finally, a disease association with a modifier gene should be explained by the functional effect of the polymorphism and demonstrate a dose-response effect.

Bile Acid Metabolism and Cellular Function

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

It is clear that bile acids play a central role in generating bile flow. Conversely, alterations in bile acid metabolism or transport can lead to hepatobiliary injury.

Bile Acid Metabolism and Inborn Errors

Inborn errors in bile acid biosynthesis are now recognized as important causes of intrahepatic cholestasis. David Russell (University of Texas Southwestern Medical Center, Dallas) described the 16 enzymes that catalyze 17 reactions involved in bile acid synthesis from cholesterol.77 Defects in several different enzymes have been associated with neonatal cholestasis (see Table 1).3, 26, 77–80 Liver disease develops as a result of both the hepatotoxicity of intermediate metabolites and secondary effects of impaired bile acid production (e.g., cholestasis and fat/fat soluble vitamin malabsorption). Therapy for these disorders is dependent on whether exogenous bile acid administration is able to supply adequate luminal concentrations of bile acids and also to inhibit the biogenesis of toxic intermediate metabolites. Animal models of these defects can be informative as to the pathophysiology of the liver disease. Targeted deletion of the 3βHSD gene seems to yield a phenocopy of human disease in mice.25

Bile Acid-Mediated Apoptosis

The mechanisms involved in liver injury associated with cholestasis have been an area of active investigation. Gregory Gores (Mayo Clinic, Rochester, Minnesota) discussed one major pathway, the induction of hepatocyte apoptosis, via either Fas or TNF-related apoptosis-inducing ligands (TRAIL).81 Caspase activation is critical for induction of apoptosis. Apoptotic hepatocytes may subsequently be engulfed by stellate cells followed by transcriptional activation of key elements involved in fibrogenesis, thus linking liver injury and fibrosis.82, 83 Liver injury is reduced in Fas death receptor-deficient mice.84 The pan-caspace inhibitor, IDN-6556, has been demonstrated to reduce hepatocyte apoptosis, to attenuate the induction of profibrogenic cytokines, and to diminish histological evidence of fibrosis in a mouse bile duct ligation model.85 Phase I investigations of this interesting approach to the treatment of liver disease have recently been reported.86

Crosstalk Between Nuclear Receptors and Cell Signaling

Saul Karpen (Texas Children's Liver Center) discussed nuclear receptors (NRs), which regulate a wide variety of critical enzymatic and transport processes in hepatocytes.87 Regulated responses to pathologic changes may occur via these NRs. Alterations in bile acid homeostasis are signaled in part through FXR.88, 89 It has been postulated that the effects of FIC1 deficiency may be mediated by alterations in the cellular localization of FXR.90 Endotoxemia and sepsis are associated with a broad range of changes in liver gene expression. The nuclear receptor RXR, the heterodimer partner of many nuclear receptors, undergoes redistribution from the nucleus to the cytoplasm in response to lipopolysaccharide treatment of mice.91 Further investigation of signaling mechanisms involved in nuclear receptor regulation of bile acid transport and synthesis will be of great relevance to human disease. Small molecular therapies may also evolve as a result of these investigations; for instance, FXR agonists may be of use in the treatment of cholestasis.92

Emerging Therapies in Cholestasis

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Over the past few years, information has been published that has helped us understand the mechanism behind the capacity of the liver to respond and adapt to cholestasis and to damage from endobiotics and xenobiotics. Among the more relevant aspects of this research is that much of the responsiveness involves activity in the hepatocyte nucleus, primarily at the level of gene transcription. Members of the NR superfamily, a group of 48 related proteins, mediate gene regulation via multiple functional domains: DNA binding, cofactor recruitment, and ligand binding. By working together, they provide a powerful means to respond to bile acid retention and metabolic demands via a coordinated activation of target gene expression. NR-regulated target genes are intimately involved in bile formation, the adaptation to cholestasis, and endobiotic/xenobiotic metabolism, suggesting that fine tuning of the response by NR ligands may well be a novel and potential means to address the inadequate hepatic response to intrahepatic cholestasis, a condition where effective therapeutics currently do not exist. The roles and responsiveness of several NR family members (RXR, FXR, PXR, CAR; see below), which are integral to basic hepatic functions (bile flow, intermediary metabolism, inflammation, and endobiotic/xenobiotic detoxification), were explored by three speakers at this Conference: Steven Kliewer, David Moore, and Stacey Jones.

Regulation of Bile Acid Metabolism by Nuclear Receptors

Steven Kliewer (University of Texas Southwestern Medical School, Dallas) provided novel insight into how one NR family member, FXR, is involved not only in regulating bile acid metabolism in the liver, but may also play a broad role in modulating the complex interrelationship between bile acids and the enterocyte response to resident bacteria in the intestinal lumen. FXR is integral to the hepatic response to cholestasis, mainly by orchestrating gene expression to unload the hepatocyte of bile acids (reducing import, increasing export).93 FXR also reduces bile acid synthesis and aids in bile acid detoxification. However, FXR is also highly expressed in ileum; the function is not readily understood. Kliewer's group, using microarray technology, found that both the natural FXR ligand, cholic acid, and a synthetic FXR agonist, GW4064, markedly activated several target genes in intestine involved in innate immunity and antimicrobial activities, including FGF15, interleukin-18, angiogenin, and inducible nitric oxide synthase. FGF15 increases Paneth cell proliferation and antimicrobial activities and is a homolog of FGF19, a known FXR target in hepatocytes. GW4064 markedly attenuated lumenal bacterial overgrowth in ileum and cecum typically seen after bile duct ligation, and this effect was completely FXR dependent. Among the more intriguing concepts that emerged from this research is that bile acids and FXR ligands may have functions that now must include the intestine in the response and pathogenesis of cholestasis.

Xenobiotic Receptors as Novel Therapeutic Targets

Two NR family members have emerged as central mediators of hepatic endobiotic and xenobiotic metabolism: CAR and PXR. These two NR family members mediate hepatoprotection by upregulating a host of target genes including cytochrome P450 family members, sulfotransferases, ligases, and transporters.94 David Moore (Baylor College of Medicine, Houston) has explored the ever-evolving and interweaving roles of these important transcriptional regulators. The emphasis has been the relative roles played by each in regulating target gene response to several hepatotoxic models, including exposure to the monohydroxylated hydrophobic bile acid, lithocholic acid (LCA). An activator of both apoptosis and cell signaling pathways, the increasing proportion of LCA in cholestatic bile has been implicated in the pathogenesis of cholestasis, yet mechanisms for its detoxification have been elusive. LCA is actually a weak activator of PXR, which may help induce its own detoxification. Moore found that CAR knockout mice are much more sensitive than PXR knockout mice to LCA-mediated hepatotoxicity. Searches for potential mediating target genes indicate that the multispecific basolateral exporter MRP3 (see Fig. 1) is upregulated by LCA treatment in wild-type and PXR knockout mice, but not in CAR knockout mice, suggesting that CAR-mediated induction of this export protein in fact may be a novel therapeutic target for unloading the hepatocyte of toxins like LCA.

Pharmacologic Modulation of Nuclear Receptors

Using three models of hepatotoxicity (bile duct ligation, CCl4, and ANIT), Stacey Jones (GlaxoSmithKline, Research Triangle Park, North Carolina) relayed information that NR ligands may reduce hepatotoxicity acting both on parenchymal and nonparenchymal cells in liver. In both the rat ANIT and BDL models, the synthetic FXR ligand GW4064 attenuated elevation in hepatic enzyme levels, but only in the ANIT model did serum bile acid and bilirubin levels decline with GW4064 treatment.95 These effects were likely related to alterations in FXR-mediated hepatoprotective pathways in hepatocytes by GW4064. Intriguingly, this FXR agonist reduced hepatic fibrosis in the CCl4 model, yet FXR targets in either Kupffer or stellate cells have yet to be described. How oral administration of GW4064 reduced hepatic collagen gene expression is not clear, but an exciting potential for therapeutics has emerged from these explorations of NR-mediated pathways.

Priorities and Opportunities in Hepatobiliary Research

  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References

Benjamin Shneider (Mount Sinai Medical Center, NY) concluded that the past decade has been an especially productive one for the investigation of the pathophysiology of intrahepatic cholestasis. Although a great deal has been accomplished, there is much more to be done, representing in part a significant portion of the priorities and opportunities in hepatobiliary research. In the era of the National Institutes of Health roadmap, it is clear that translationally oriented areas of investigation are of high priority. Many of the concepts are described in the National Institutes of Health Action Plan for Liver Disease Research (

A wide range of genes have been identified to underlie the pathogenesis of cholestatic liver diseases, including many that are described earlier in this review. Fully one third of children with low GGT familial intrahepatic cholestasis have no demonstrable genetic defect in FIC1 or BSEP. Thus, one of the great opportunities that exists is the potential identification of new genes that are involved in the pathogenesis of cholestatic liver disease (Aagenaes syndrome [LCS1, 15q,],96 Jeune's syndrome [15q13],97 Rotor's syndrome, etc.).

A significant percentage of patients with cholestatic liver disease have no clear underlying cause, so it is likely that continued mining for new genetic defects will be fruitful. Identification of a gene defect that underlies a specific liver disease is only the first step in determining the molecular pathophysiology of that disorder. Clearly elucidating the pathophysiologic relationship of defects in a specific gene to a particular disease is a high priority for future investigations. In light of the large number of newly identified genes, there are great opportunities in this area of investigation. There are many options for assessing the role of a specific gene in the pathogenesis of disease, although a priori it is difficult to know which approach will be most fruitful. Possible approaches include comprehensive analyses of genotype/phenotype relationships, modeling of genetic disorders in cell culture, isolated cell units (e.g., hepatocytes or bile ducts) and/or knockin/knockout animal models.

Direct translation of recent molecular advances to the diagnosis and treatment of human disease is both an obligation and high priority for the research community. It is critically important to be able to make specific molecular diagnoses in patients with liver disease. Standard genotype analysis in large groups of patients is not practical. Therefore, development of gene chip approaches is a priority. In addition, biomarkers for genetic defects need to be explored and/or identified (e.g., serum lipoprotein X or biliary phospholipid for MDR3 defects or “Byler's bile” for FIC1 defects). Development of well-characterized and widely available molecular reagents for investigation of human tissue samples is both in great demand and an important opportunity. Clinical investigation of the relationship between genotype and therapeutic response is a major priority for future research. Identification of patients with diseases that are amenable to medical (partially functional MDR3 defects and ursodeoxycholic acid therapy) or nontransplant surgical therapy (FIC1 or BSEP and partial biliary diversion/ileal exclusion) is extremely important, especially in light of the fact that liver transplantation is associated with a low but real risk of death. In addition, liver transplantation seems to be only partially effective in diseases like FIC1 deficiency, where the manifestations of disease are systemic. The effective use of small molecules or targeted drug therapy for the treatment of specific genetic liver diseases is one of the most important priorities for future investigation.

The rapid increase in our understanding of the molecular basis of pediatric liver diseases provides ample opportunities for productive and rewarding investigations of hepatobiliary disease. The highest priority for investigation will be in the context of translationally relevant inquiry. Funding of the Biliary Atresia Research Consortium ( and the affiliated Cholestatic Liver Disease Consortium ( will provide an important infrastructure and mechanism for many of these future investigations. We look forward to continued progress in understanding disorders of intrahepatic cholestasis.


  1. Top of page
  2. Intrahepatic Cholestasis: From the Bedside to the Bench
  3. Molecular Basis of Canalicular Transport
  4. Molecular Defects as the Basis of Cholestatic Syndromes
  5. Developmental Abnormalities of the Biliary System
  6. Insight From Global Genomics and Phenotypic Expression
  7. Bile Acid Metabolism and Cellular Function
  8. Emerging Therapies in Cholestasis
  9. Priorities and Opportunities in Hepatobiliary Research
  10. Acknowledgements
  11. References
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