Physiol Rev
83: 633– 671, 2003; 10.1152/physrev.00027.2002.
Bile Salt Transporters: Molecular Characterization,
Function, and Regulation
MICHAEL TRAUNER AND JAMES L. BOYER
Division of Gastroenterology and Hepatology, Department of Internal Medicine, Karl-Franzens University,
School of Medicine, Graz, Austria; and Department of Medicine and Liver Center, Yale University
School of Medicine, New Haven, Connecticut
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I. Normal Physiology of Bile Salt Transport
A. Basic Principles of Bile Secretion and Bile Salt Transport
B. Hepatocellular Bile Salt Transport
C. Cholangiocellular Bile Salt Transport
D. Intestinal Bile Salt Transport
E. Renal Bile Salt Transport
F. Placental Bile Salt Transport
II. Regulation of Bile Salt Transporters in Normal Physiology
A. Transcriptional Regulation of Bile Salt Transporters
B. Posttranscriptional Regulation of Bile Salt Transporters
III. Bile Salt Transport Defects in Cholestatic Liver Injury
A. Genetic Defects in Bile Salt Transport Proteins (FIC1, BSEP, MRP2) and MDR3
B. Acquired Defects in Bile Salt Transport Proteins in Cholestasis
IV. Liver Regeneration
V. Summary
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Trauner, Michael, and James L. Boyer. Bile Salt Transporters: Molecular Characterization, Function, and
Regulation. Physiol Rev 83: 633– 671, 2003; 10.1152/physrev.00027.2002.—Molecular medicine has led to rapid
advances in the characterization of hepatobiliary transport systems that determine the uptake and excretion of bile
salts and other biliary constituents in the liver and extrahepatic tissues. The bile salt pool undergoes an enterohepatic circulation that is regulated by distinct bile salt transport proteins, including the canalicular bile salt export
pump BSEP (ABCB11), the ileal Na⫹-dependent bile salt transporter ISBT (SLC10A2), and the hepatic sinusoidal
Na⫹- taurocholate cotransporting polypeptide NTCP (SLC10A1). Other bile salt transporters include the organic
anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3
(ABCC2,3). Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta.
Expression of these transport proteins is regulated by both transcriptional and posttranscriptional events, with the
former involving nuclear hormone receptors where bile salts function as specific ligands. During bile secretory
failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile
salt retention and which facilitate extrahepatic routes of bile salt excretion. This review is a comprehensive
summary of current knowledge of the molecular characterization, function, and regulation of bile salt transporters
in normal physiology and in cholestatic liver disease and liver regeneration.
I. NORMAL PHYSIOLOGY OF BILE SALT
TRANSPORT
A. Basic Principles of Bile Secretion
and Bile Salt Transport
Bile is a vital secretion, essential for intestinal digestion and absorption of lipids. Moreover, bile is an important route of elimination for environmental toxins, carcinwww.prv.org
ogens, drugs, and their metabolites (xenobiotics). Bile is
also the major route of excretion for endogenous compounds and metabolic products (endobiotics) such as
cholesterol, bilirubin, and hormones (44, 262). Bile is
primarily secreted by hepatocytes into minute channels
arranged as a meshwork of tubules or canaliculi between
adjacent hepatocytes. “Canalicular bile” accounts for
⬃75% of daily bile production in humans and is modified
by secretory and absorptive processes as it passes along
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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MICHAEL TRAUNER AND JAMES L. BOYER
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by the liver are filtered at the glomerulus and excreted
into urine, where they are reabsorbed by transporters in
the brush border of the proximal convoluted tubule (386).
Most hepatic transport systems are not fully expressed until shortly before/after birth (19). Thus the
fetus must rely entirely on elimination of “biliary” constituents via the placenta and maternal liver. This “placentamaternal liver tandem” is responsible for protecting the
fetal organism from potentially harmful compounds.
Recent cloning studies have characterized the molecular properties of most of the hepatobiliary transport
systems required for uptake and excretion of bile salts
and other biliary constituents in liver and extrahepatic
tissues (Table 1, Fig. 1). These studies have done much to
advance our understanding of the molecular basis of bile
formation (205, 210, 245, 258, 259, 342, 358, 359). However, bile formation normally depends not only on the
proper function of these transport systems, but also on an
intact cytoskeleton required for the movement of vesicles
and bile canalicular contractions, and on junctional contacts that seal off the bile canaliculi and maintain cell
polarity. Signal transduction mechanisms also regulate
and coordinate these various processes. Although this
review is focused on the molecular mechanisms involved
in bile salt transport in the liver and the enterohepatic
circulation, the interested reader may wish to consult
other reviews that address these cellular mechanisms (15,
44, 98, 250, 251, 286).
B. Hepatocellular Bile Salt Transport
The hepatocyte is a polarized epithelial cell with
basolateral (sinusoidal) and apical (canalicular) plasma
membrane domains. Bile salts are concentrated in bile by
active transport systems that are arranged in a polarized
fashion (Table 1, Fig. 1). Hepatic uptake of biliary constituents (and their precursors) is initiated at the basolateral membrane, which is in direct contact with portal
blood plasma via the fenestrae of the sinusoidal endothelial cells and the space of Disse. After uptake into hepatocytes, bile salts and other cholephiles reach the canalicular pole by diffusion either in the aqueous cytoplasm
or within intracellular lipid membranes, depending on
their hydrophobicity. The canalicular membrane represents the excretory pole of the hepatocyte and forms the
border of the bile canaliculus. Canalicular excretion of
biliary constituents represents the rate-limiting step of
bile secretion since biliary constituents are excreted
against high concentration gradients into bile. The basolateral and canalicular membranes differ in their biochemical composition and functional characteristics and
are separated by tight junctions that seal off the bile
canaliculi and hence form the only anatomical barrier
maintaining the concentration gradients between blood
and bile (38, 44).
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the bile ductules and ducts. The quantity of “ductular/
ductal bile” varies with the species and responses to
enteric hormonal stimuli and varies from as little as 5% in
rats to as much as 25– 40% of secretion in humans. Bile is
further concentrated up to 10-fold in the gallbladder before reaching the intestine except for species like the rat
where the gallbladder is absent (44, 262).
Bile secretion is an osmotic process driven predominantly by active excretion of organic solutes into the bile
canaliculus, followed by passive inflow of water, electrolytes, and nonelectrolytes (e.g., glucose) from hepatocytes and across semipermeable tight junctions (42, 332).
The main organic solutes of bile are bile salts, phospholipids, and cholesterol, which form mixed micelles in bile.
The vectorial excretion of bile salts from blood into bile
represents the major driving force for bile flow (“bile
salt-dependent” bile flow). The human bile salt pool size is
⬃50 – 60 ␮mol/kg body wt and averages 3– 4 g. Most of the
bile salt pool is stored in the gallbladder during the fasting
state (139, 210, 245). Canalicular excretion of reduced
glutathione (GSH) and bicarbonate (HCO⫺
3 ) constitute the
major components of the “bile salt-independent” fraction
of bile flow. However, HCO⫺
3 secretion occurs mainly at
the level of bile duct epithelial cells (cholangiocytes), in
response to stimulation by hormones and neuropeptides
such as secretin, vasoactive intestinal peptide, bombesin,
etc. (22, 39, 167).
Many organic biliary constituents including bile salts,
cholesterol, and phospholipids are reabsorbed with high
efficiency after reaching the small intestine and recirculate via the portal blood back to the liver. Thus the human
bile salt pool circulates 6 –10 times per day, resulting in a
daily bile salt excretion of 20 – 40 g. Despite a high degree
of intestinal bile salt conservation, 0.5 g of bile salts are
lost through fecal excretion and must be replaced by de
novo bile salt synthesis, which thus contributes to only 3–5%
of the bile salts that are excreted into bile (139, 210, 245).
Bile salts may also undergo “cholehepatic shunting”
from the bile duct lumen, via cholangiocytes and the
periductular capillary plexus. Bile salt reabsorption by
cholangiocytes may contribute in part to the conservation
of bile salts and the generation of a hypercholeretic bile
flow (123, 395). Although this pathway probably plays a
minor role under normal physiological conditions, cholehepatic shunting of bile salts may become an important
escape route for bile salts under cholestatic conditions
when the bile duct epithelium proliferates. Moreover, bile
salt uptake into cholangiocytes and gallbladder epithelial
cells may also play an important role for cell signaling in
the regulation of secretory and proliferative events within
the biliary tree (7, 10, 67).
Bile salts are efficiently removed from portal blood
by the liver via high-affinity, low Michaelis constant (Km)
transporting polypeptides in the basolateral sinusoidal
membrane. Bile salts that escape the first-pass clearance
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BILE SALT TRANSPORTERS
TABLE 1. Nomenclature, location, and function of the major hepatobiliary membrane transporters
in liver and extrahepatic tissues
Name
Abbreviation
(Gene Nomenclature)
Function
Basolateral membrane of hepatocyte
Sodium-taurocholate
cotransporting polypeptide
Organic anion transporting
proteins
Multidrug resistance-associated
protein 3*
NTCP (SLC10A1)
OATPs (SLC21A)
MRP3 (ABCC3)
Canalicular membrane of hepatocyte
Multidrug resistance-1
P-glycoprotein*
Multidrug resistance-3
P-glycoprotein (phospholipid
transporter)*
Multidrug resistance-associated
protein-2 (Canalicular
conjugate export pump)*
Canalicular bile salt-export
pump*/(sister of Pglycoprotein)
Familial intrahepatic
cholestasis-1
Chloride-bicarbonate anion
exchanger isoform-2
MDR1 (ABCB1)
MDR3 (ABCB4)
MRP2 (ABCC2)
BSEP (SPGP)
(ABCB11)
FIC1 (ATP8B1)
AE2 (SLC4A2)
ATP-dependent excretion of various organic cations, xenobiotics, and cytotoxins
into bile
ATP-dependent translocation of phosphatidylcholine from inner to outer leaflet of
membrane bilayer; the phospholipid export pump
Mediates ATP-dependent multispecific organic-anion transport (e.g., bilirubin
diglucuronide, sulfates, glutathione conjugates) into bile; major determinant of
bile salt-independent bile flow by GSH transport
ATP-dependent transport of monovalent bile salts into bile; stimulates bile salt
dependent bile flow
Potential aminophospholipid translocating ATPase (function not known); gene
defect in Byler’s disease
Acid loader. Excretes bicarbonate into bile and stimulates bile salt-independent
bile flow
Cholangiocytes
Ileal (apical) sodium-dependent
bile salt transporter
ISBT (ABST)
(SLC10A2)
Cystic fibrosis transmembrane
regulator
Chloride-bicarbonate anion
exchanger isoform 2
Multidrug resistance-associated
protein 3*
CFTR (ABCC7)
AE2 (SLC4A2)
MRP3 (ABCC3)
Located on the luminal membrane; functions to remove bile salts from bile; may
facilitate bile salt removal during cholestasis; identical gene product to ileal
transporter
Chloride channel on the luminal membrane; facilitates chloride entry into bile for
exchange with HCO⫺
3 anions; mutations in CFTR can result in cholestasis
Located on luminal membrane; facilitates bicarbonate secretion into bile and
contributes to bile flow independent of bile salts
Expressed on basolateral membrane of cholangiocyte; may be major transporter
for returning bile salts to portal circulation from bile during obstructive
cholestasis
Intestine
Organic anion transporting
polypeptide 3
Ileal sodium dependent bile
salt transporter
Multidrug resistance-associated
protein 3*
Oatp3 (Slc21a7)
Sodium-independent uptake of bile salts from intestinal lumen
ISBT (ABST)
(SLC10A2)
MRP3 (ABCC3)
Primary carrier for sodium-dependent bile salt uptake from intestine by ileum;
critical determinant of enterohepatic circulation of bile salts
Expressed on basolateral membrane of ileum and colon; may be major transporter
for returning bile salts to portal circulation
Kidney
Ileal sodium dependent bile
salt transporter
ISBT (ABST)
(SLC10A2)
Multidrug-resistance-associated
protein 2.*
MRP2 (ABCC2)
GSH, reduced glutathione.
Located on the luminal membrane of the proximal tubule; functions to take up bile
salts from the glomerular filtrate; downregulated in cholestasis to facilitate bile
salt excretion in urine; identical gene product to the ileal transporter
Located on the luminal membrane of the proximal tubule, this transporter is an
ATP-dependent multispecific organic anion transporter for divalent organic anion
conjugates (sulfates, glucuronide, and glutathione conjugates) into urine; this gene
product is identical to the canalicular conjugate export pump and is postulated to
function to facilitate renal excretion of bile salt conjugates in cholestasis
* These transporters are members of the ATP-binding cassette family.
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Primary carrier for sodium-dependent conjugated bile salt uptake from portal
blood
Multispecific organic anion transporters for sodium-independent uptake of bile
salts and a broad range of other organic anions and cations
Multispecific organic solute transporter, weakly expressed in normal liver but
highly upregulated on basolateral membrane of hepatocytes in cholestasis;
capable of extruding bile salt conjugates from liver
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MICHAEL TRAUNER AND JAMES L. BOYER
1. Basolateral bile salt uptake
Basolateral bile salt transport systems are essential
for bile formation since ⬃95% of bile salts excreted into
bile by the liver are reabsorbed on each pass through the
intestine and undergo an “enterohepatic circulation”
(139). This process is highly efficient with first-pass extraction rates of conjugated bile salts in the range of
75–90% depending on bile salt structure and is independent of systemic bile salt concentrations (241). Unconjugated bile salts are weak acids that are uncharged at the
physiological pH in plasma and thus can traverse cell
membranes by passive diffusion. However, taurine or glycine conjugated bile acids require an active transport
mechanism for cellular uptake (241). Hepatic uptake of
bile salts occurs against a 5- to 10-fold concentration
gradient between the portal blood plasma and the hepatocyte cytosol and is mediated by both sodium-dependent
and -independent mechanisms (262). Most substances
cleared by the liver (including bile salts) are highly protein bound to serum albumin. Because only the unbound
fraction of biliary constituents enter the liver, the ligand
must dissociate upon contact with the sinusoidal membrane of the hepatoctye. Therefore, efficient extraction
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from albumin is an important step (293). Because simple
dissociation from serum albumin is too slow (100, 382),
this dissociation must be facilitated at the cell membrane,
although proof for an hepatocellular albumin receptor is
lacking (176, 271, 383). Under physiological conditions,
bile salts are removed from sinusoidal blood predominantly by zone 1 (periportal) hepatocytes (119, 159).
Hepatocytes in zone 3 downstream are only recruited at
higher bile salt loads (215), such as may occur postprandially or under cholestatic conditions. Thus the normal
liver has considerable functional reserve capacity for bile
salt uptake. There are several different transport systems
involved in hepatic bile salt uptake, and these include a
high-affinity Na⫹-dependent bile salt transporter Ntcp/
NTCP (Slc10a1/SLC10A1) and a family of multispecific
organic anion transporters (Oatps/OATPs; Slc21a/
SLC21A) that can mediate Na⫹-independent bile salt uptake.
A) Sodium-dependent uptake via Ntcp/NTCP. The
Na⫹-dependent pathway accounts for ⬎80% of conjugated taurocholate uptake but ⬍50% of unconjugated
cholate uptake (210, 245). Because most bile salts are
conjugated, the Na⫹-dependent transport system Ntcp/
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⫺
FIG. 1. Hepatobiliary transport systems in liver and extrahepatic tissues in humans. Bile salts (BS ) are taken up by
hepatocytes via the basolateral Na⫹/taurocholate cotransporter (NTCP) and organic anion transporting proteins
(OATPs). Monovalent BS⫺ are excreted via the canalicular bile salt export pump (BSEP) while divalent BS⫺ together
with anionic conjugates (OA⫺) are excreted via the canalicular conjugate export pump (MRP2). The phospholipid export
pump (MDR3) facilitates excretion of phosphatidylcholine (PC), which forms mixed micelles in bile together with BS⫺
and cholesterol. Cationic drugs (OC⫹) are excreted by the multidrug export pump (MDR1). Other basolateral isoforms
of the multidrug resistance-associated protein (MRP1 and MRP3) provide an alternative route for the elimination of BS⫺
and nonbile salt OA⫺ from hepatocytes into the systemic circulation. BS⫺ are reabsorbed in the terminal ileum via ileal
Na⫹-dependent bile salt transporter (ISBT) and effluxed by MRP3. Similar mechanisms exist in proximal renal tubules
and cholangiocytes where an additional, truncated isoform (t-ISBT) may be involve in BS⫺ efflux from cholangiocytes.
MRP2 is also present in the apical membrane of enterocytes and proximal renal tubules, while MDR1 is also found in
intestine and bile ducts.
BILE SALT TRANSPORTERS
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ids and shares 77% amino acid identity with rat liver Ntcp.
The functional properties of NTCP are very similar to rat
Ntcp, although it has a higher affinity for taurocholate
than the rat and mouse orthologs (125). The NTCP gene
has been mapped to chromosome 14q24.1–24.2 (210, 245).
Microsomal epoxide hydrolase (mEH) has also been
proposed to mediate Na⫹-dependent bile salt uptake (365,
366, 399), including a microsomal isoform and an isoform
localized to the basolateral membrane of hepatocytes.
Expression of the cell-surface isoform of mEH in MadinDarby canine kidney (MDCK) cells transfers/mediates
Na⫹-dependent DIDS-inhibitable taurocholate transport
(366). Although mEH could mediate Na⫹-dependent
cholate uptake in hepatocytes, it has to be considered that
⬎50% of cholate uptake is Na⫹ independent, and Ntcp
also contributes to Na⫹-dependent cholate uptake (210).
Moreover, mEH (⫺/⫺) mice have no abnormalities in bile
salt homeostasis, suggesting that mEH is not an important
determinant for physiological bile salt transport (253).
The microsomal isoform of mEH could be involved in
vesicular bile salt transport from the basolateral to the
canalicular membrane, but the physiological significance
of this pathway is not clear (11).
Taken together, the current findings suggest that the
features of Na⫹-dependent bile salt uptake into hepatocytes can be largely explained by the molecular properties
of Ntcp/NTCP.
B) Sodium-independent uptake via Oatps/OATPs. In
contrast to Na⫹-dependent bile salt uptake, Na⫹-independent uptake of bile salts is determined by several different
gene products. Oatps/OATPs are multispecific transporter
systems with a wide substrate preference for mostly amphipathic organic compounds, including conjugated and
unconjugated bile salts, bromosulfophthalein (BSP), bilirubin, DIDS, cardiac glycosides and other neutral steroids, linear and cyclic peptides, mycotoxins, selected
organic cations, and numerous drugs such as pravastatin
(for review, see Refs. 210, 242, 245). Na⫹-independent bile
salt uptake is quantitatively less significant than sodiumdependent uptake and appears to be largely mediated by
facilitated exchange with intracellular anions (e.g., GSH,
HCO⫺
3 ) (210).
In rat liver, basolateral sodium-independent bile salt
uptake is mediated by three members of the organic anion
transporting protein (Oatp/OATP) family. Other members
of the Oatp/OATP family do not appear to be bile salt
carrier systems. Oatp1 (Slc21a1) from rat liver was the
first cloned member of the Oatp family and is a 670-amino
acid glycoprotein with an apparent molecular mass of 80
kDa and 12 putative transmembrane-spanning domains
(152). Oatp1 is localized to the basolateral membrane of
hepatocytes, the apical membrane of kidney proximal
tubular cells, and choroid plexus epithelial cells (16, 28).
Developmentally, Oatp1 expression precedes expression
of Ntcp, and its mRNA can be detected already at day 16
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NTCP is the most relevant Na⫹-dependent bile salt uptake
system. Bile salt uptake via Ntcp/NTCP is unidirectional
with a sodium-to-taurocholate stoichiometry of 2:1, i.e.,
cotransport of two Na⫹ with one taurocholate molecule,
and electrogenic, i.e., it is driven by both the transmembrane Na⫹ gradient which in turn is maintained by a
Na⫹-K⫹-ATPase and the intracellular electrical potential
derived from the outward diffusions of K⫹ (210, 245).
The basolateral Na⫹-dependent bile salt transporter,
the Na⫹-taurocholate cotransporting polypeptide (Ntcp/
NTCP), has been cloned from rat, human, mouse, and
rabbit liver (57, 125, 127, 198). The rat liver Ntcp (gene
symbol Slc10a1) consists of 362 amino acids with an
apparent molecular mass of 51 kDa, 2 NH2-terminal glycosylation sites, and 7 putative transmembrane domains
(14, 127, 337), the latter being unique among sodium
cotransporters (389). The Ntcp gene is located on rat
chromosome 6q24 and spans 16.5 kb, with five exons
separated by four introns (210, 245). Ntcp is localized
exclusively at the basolateral membrane of differentiated
mammalian hepatocytes and is hepatocyte specific (14,
337). Isolation of hepatocytes results in rapid reduction of
Ntcp expression and loss of hepatocellular bile salt uptake in vitro (295). Ntcp is distributed homogeneously
throughout the liver lobule/acinus (337). During rat development, Ntcp can first be detected between days 18 and
21 of gestation (43). Functionally, Ntcp transports all
physiological bile salts (e.g., as taurocholate, glycocholate, taurochenodeoxycholate, tauroursodeoxycholate)
when expressed in oocytes and various cell systems, although its transport activity is highest for glycine- and
taurine-conjugated dihydroxy and trihydroxy bile salts
(210, 242, 245). Antisense experiments in Xenopus leavis
oocytes injected with total rat liver mRNA have revealed
a 95% reduction of Na⫹-dependent taurocholate transport
(126), suggesting that Ntcp is the major if not the only
Na⫹-dependent bile salt uptake system in rat liver. Although bile salts are the major physiological substrate for
Ntcp, other compounds such as estrogen conjugates (estrone-3-sulfate), bromosulfophthalein, dehydroepiandrosterone sulfate, thyroid hormones, and the drug ONO-1301
can also be transported (210, 242, 245, 349). In addition,
Ntcp also mediates uptake of drugs that are covalently
bound to taurocholate such as chlorambucil-taurocholate
(206).
In mouse liver, two alternatively spliced Ntcp isoforms (Ntcp1, Slc10a1; Ntcp2, Slc10a2) have been
cloned. Ntcp is a 362-amino acid protein, while Ntcp2 is a
truncated 317-amino acid protein produced by alternative
splicing (57). Both Ntcps mediate Na⫹-dependent taurocholate uptake when expressed in oocytes. However,
mRNA levels of Ntcp2 are ⬃50-fold lower than Ntcp1, and
its functional relevance under normal and pathological
conditions remains to be determined (57).
Human NTCP (SLC10A1) consists of 349 amino ac-
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MICHAEL TRAUNER AND JAMES L. BOYER
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tein. Oatp2 is localized to the basolateral membrane of
hepatocytes, retina, endothelial cells of the blood-brain
barrier, and the basolateral membrane of choroid plexus
epithelial cells (105, 292, 331). While Oatp1 is homogeneously distributed throughout the liver lobule as revealed by in situ hybridization and immunofluorescence
studies, Oatp2 is predominantly expressed in perivenous/
pericentral hepatocytes with sparing of the innermost one
to two cell layers (163, 292). Induction of Oatp2 expression by phenobarbital results in a homogeneous distribution throughout the liver (128). Because the major proportion of hepatocellular bile salt uptake normally occurs
in the periportal region, Oatp1 is mainly implicated in
Na⫹-independent bile salt uptake under normal conditions, while pericentral hepatocytes may become recruited only under cholestatic conditions with downregulation of Ntcp and Oatp1 and spillover of unextracted bile
salts form the periportal to the pericentral region (51,
220). The substrate specificity of Oatp2 is similar, but not
identical, to that of Oatp1, with similar affinities for the
bile salts taurocholate and cholate (203, 210, 242, 245).
These include taurocholate, cholate, glycocholate, taurochenodeoxycholate, tauroursodeoxycholate, DHEAS, estradiol 17␤-glucuronide, T3 and T4, ouabain, digoxin, pravastatin, the endothelin antagonist BQ-123, the opioid receptor antagonists D-penicillamine-enkephalin and Leuenkephalin, biotin, fexofenadine, and the bulky type II
organic cations APD-ajmalium and recuronium. Importantly, in contrast to Oatp1, bilirubin monoglucuronide,
BSP, LTC4, and Gd-EOB-DTPA (gadoxetate) are not transported via Oatp2 (203, 210, 242, 245). Moreover, Oatp2
transports ouabain with higher affinity and is unique in
mediating high-affinity transport of the cardiac glycoside
digoxin (263). Hence, Oatp1 appears to prefer amphipathic organic anions, whereas Oatp2 has an extended
substrate preference for neutral compounds (203, 210,
245). Similar to Oatp1, the driving force for Oatp2-mediated uptake appears to be exchange with GSH and GSH
conjugates (228).
The third Oatp family member involved in hepatic
bile salt uptake in rat liver is Oatp4 (Slc21a10). Oatp4 is
the full-length isoform of the rat liver-specific transporter
1 (rLst-1) (58). Although three rLst-1 splice variants have
been detected (70), full-length Oatp4 represents quantitatively and functionally the most relevant protein in rat
liver (58). Compared with rLst-1, Oatp4 is more highly
expressed in liver and contains an additional 35 amino
acids, resulting in a predicted 12 transmembrane topology
characteristic of all Oatps (58). Oatp4 shares 43% amino
acid identity with Oatp1 and 44% identity with Oatp2 (210,
245). While rLst-1 transports only taurocholate, Oatp4
also transports BSP, estrone-3-sulfate, estradiol 17␤-glucuronide, DHEAS, prostaglandin E2, LTC4, the thyroid hormones T3 and T4, and gadoxetate (58). Similar to Oatp1
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of gestation in the developing rat liver (91). Oatp1 is a
highly versatile and multispecific transport system that
transports a wide variety of amphipathic substrates with
differing affinities, including bile salts (although with
lower affinities than Ntcp), nonbile salt organic anions,
organic cations, neutral steroids, and small peptides.
More specifically, Oatp1 transports bile salts (taurocholate, cholate, glycocholate, taurochenodeoxycholate,
tauroursodeoxycholate, taurodeoxycholate, taurohyodeoxycholate), organic anionic dyes such as BSP, bilirubin
monoglucuronide, thyroid hormones including triiodothyronine (T3) and thyroxine (T4), steroid hormones (aldosterone, dexamethasone, cortisol) and steroid conjugates
[17␤-estradiol glucuronide, estrone-3-sulfate, dehydroepiandrosteronsulfate (DHEAS)], leukotriene C4 (LTC4) and
other glutathione conjugates (dinitrophenylglutathione),
the anionic magnetic resonance imaging contrast agent
gadoxetate (Gd-EOB-DTPA, or gadolinium-ethoxybenzyldiethylenetriamine-pentacetic acid), neutral steroids such
as ouabain, the dipeptidic angiotensin-converting enzyme
inhibitors enalapril and temocaprilat, the HMG-CoA reductase inhibitor pravastatin, the peptidomimetic thrombin inhibitor CRC 220, the endothelin receptor antagonist
BQ-123, the opioid receptor antagonists D-penicillamineenkephalin and deltorphin II, the antihistamine fexofenadine, the mycotoxin ochratoxin A, and even organic cations (amphipathic “bulky” type II organic cations) such as
N-propylajmalinium and APD-ajmalinium, and to a lesser
degree methyl-quinine and recuronium (203, 210, 242, 244,
245). More recently, sulfolithotaurocholate has been identified as a substrate for Oatp1 and Oatp2 (247, 292).
Despite this wide substrate specificity, Oatp1 does not
mediate uptake of unconjugated bilirubin (210). Antisense
studies in oocytes injected with total rat liver mRNA have
revealed that Oatp1 can account for 80% of Na⫹-independent taurocholate uptake but for only 50% of BSP uptake
of rat liver, consistent with the existence of additional
Oatps (126). Oatp1 appears to function as an anion exchanger with HCO⫺
3 (303) and/or GSH (227) as counteranions. Thus continuous GSH efflux from hepatocytes
along its in-to-out gradient could provide an efficient driving force for Oatp1-mediated substrate uptake into hepatocytes. Mouse Oatp1 (Slc21a1) has the same substrate
specificity as rat Oatp1 and is expressed predominantly in
liver with additional expression in kidney (124). Mouse
Oatp1 gene has been mapped to chromosome XA3-A5
(124).
Based on homology with Oatp1, a second Oatp2
(Slc21a5) was cloned originally from a rat brain cDNA
library. Oatp2 is also highly expressed in liver and kidney
(263). Oatp2 is a 661-amino acid protein with an apparent
molecular mass of 92 kDa and is 77% identical to Oatp1
(263, 292). Like Oatp1, Oatp2 also has 12 putative transmembrane spanning domains and represents a glycopro-
BILE SALT TRANSPORTERS
Physiol Rev • VOL
the basolateral membrane of hepatocytes (2, 142, 189).
OATP-C exhibits the highest amino acid identity (64%)
with rat Oatp4 (210). As a result of gene duplication in
humans, both OATP-C and OATP-8 represent the human
orthologs of rodent Oatp4. OATP-C transports taurocholate (with slightly lower affinity than NTCP), bilirubin
monoglucuronide, conjugated steroids, eicasonoids, thyroid hormones, and peptides (210). The substrate specificity of OATP-C is very comparable to rat Oatp4 and,
more specifically, includes taurocholate, bilirubin monoglucuronide, BSP, estrone-3-sulfate, estradiol 17␤-glucuronide, DHEAS, prostaglandin E2, thromboxane B2,
LTC4 and LTE4, the thyroid hormones T3 and T4, and
pravastatin (210). Although OATP-C exhibits large overlapping substrate specificities with other OATPs of human liver (208), a unique property is its capacity to transport unconjugated bilirubin (77, 78). In contrast to OATPA, OATP-C shows a substrate preference for organic
anions and does not include amphipathic organic cations.
Of note, OATP-C transports taurocholate with slightly
lower affinity than NTCP, although current data suggest
that it represents an important Na⫹-independent bile salt
uptake system in human liver (208, 210). Polymorphisms
in OATP-C associated with variable degrees in plasma
membrane expression may represent an important factor
influencing drug disposition (354). In addition, a recently
identified OATP-C mutation severely interferes with normal basolateral OATP-C expression and function (J.
König and D. Keppler, personal communication).
OATP8 (SLC21A8) is 80% identical with OATP-C and
is also expressed at the basolateral membrane of human
hepatocytes (190). Thus OATP-C and -8 are expressed in
a liver-specific fashion. The data on bile salt transport by
OATP8 are controversial since bile salts were not transported when OATP8 was expressed in mammalian cells
(190), but they were when OATP8 was expressed in oocytes (208, 210). Interestingly, OATP8 (and rat Oatp4) has
been identified as hepatic uptake systems for cholecystokinin (148).
OATP-B (SLC 21A9) is also expressed at the basolateral membrane of human hepatocytes but does not
transport bile salts (208, 351). Although OATP-B is predominantly expressed in liver, it is not liver specific and is
found in many other tissues (208) including placenta (341)
(see sect. IF).
Hence, although sodium-independent bile salt transport is an intrinsic feature of several Oatps/OATPs, each
of these polyspecific transport systems exhibits additional substrate specificities and preferences that may
even be specific for certain substrates (e.g., digoxin for
Oatp2 and OATP8). For many of these substrates, Oatp/
OATP-mediated uptake appears to be the main if not the
only uptake route, based on the similarities of Km values
between oocyte expression systems and total liver.
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and Oatp2, Oatp4 is a multispecific transporter with high
affinities for BSP, DHEAS, LTC4, and anionic peptides. In
addition, Oatp4 appears to be particularly involved in the
hepatic clearance of anionic peptides including microcystin (a toxin derived from algae) and cholecystokinin (a
gastrointestinal peptide hormone) (210, 245).
Oatp3 (Slc21a7) is not expressed in liver in contrast
to initial reports (1) but may be important for intestinal
bile salt uptake (370) (see sect. ID). Oatp3 has a similar
broad substrate specificity but much lower affinities than
Oatp4 (59). Thus, while Oatp4 works in concert with
Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the
small intestine (59).
An evolutionary ancient liver specific Oatp has been
characterized in a primitive vertebrate, the small skate
(Raja erinacea) with substrate similarities to rat Oatp4
and human OATP-C (55). This Oatp shares only ⬃40 –50%
amino acid identity with other liver-specific OATPs/Oatps
and is most closely related to human OATP-F in brain.
These findings emphasize the early evolutionary development of the Slc21a/SLC21A transporter family.
Taken together, the transport characteristics of
Oatp1, -2, and -4 can account for the bulk of Na⫹-independent bile salt uptake in liver (210, 245).
In humans, three liver-specific OATPs (OATP-A,
OATP-C, and OATP-8) transport bile salts (208). For bile
salt uptake, OATP-C is the most relevant isoform (210,
245).
The first human OATP identified in human liver was
OATP-A (SLC21A3) (207). It consists of 670 amino acids
and exhibits only a 67% amino acid homology to rat Oatp1
(which is not an ortholog as suggested by low amino acid
identity and different substrate specificity), 73% with
Oatp2, 42% with Oatp4, and 44% with human OATP-C.
Although OATP-A was originally cloned from human liver
(207), its hepatic expression level is low, and its contribution to overall hepatic bile salt uptake is probably
minor (245). It is predominantly expressed in human cerebral endothelial cells where it may play a role in the
blood-brain barrier (104). OATP-A transports bile salts
such as taurocholate, cholate, tauroursodeoxycholate, in
addition to BSP, estrone-3-sulfate, DHEAS, the magnetic
resonance imaging contrast agent Gd-B 20790, the opioid
receptor antagonists D-penicillamine-enkephalin, and deltorphin II, fexofenadine, and the bulky type II organic
cations APD-ajmalium, recuronium, N-methylquinine, and
N-methylquinidine. Transport rates were highest for the
organic cation N-propylajmalium and the peptidomimetic
drug CRC 220. In addition, OATP-A mediates unique highaffinity transport of the bulky lipophilic organic cations
methylquinine and methylquinidine (210, 245).
OATP-C (SLC21A6) also known as LST-1 and OATP2
consists of 691 amino acids and is selectively expressed at
639
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MICHAEL TRAUNER AND JAMES L. BOYER
2. Basolateral bile salt efflux
Physiol Rev • VOL
3. Intracellular bile salt transport
The mechanisms involved in the transcellular movement of bile salts across hepatocytes from the basolateral
to the canalicular membrane are poorly understood. With
physiological bile salt loads, a considerable fraction is
bound to intracellular binding proteins (e.g., glutathioneS-transferases, 3-hydroxysteroid dehydrogenase, fatty acid-binding proteins in rat liver; a 36-kDa bile acid binding
protein in human liver) and diffuses to the canalicular
membrane in a protein bound form (for review, see Ref.
4). In addition, free, unbound intracellular bile salts may
reach the canalicular membrane by rapid diffusion. At
high bile salt loads, increasing partition of hydrophobic
bile salts occurs into intracellular organelles including
endoplasmatic reticulum, Golgi apparatus, and other
membrane-bound compartments (4). Early reports of a
direct vesicle-dependent fraction of transcellular bile salt
transport can now be better explained by vesicular targeting of their respective transport systems (183).
4. Canalicular bile salt excretion
Canalicular bile secretion represents the rate-limiting
step in bile formation. The canalicular membrane contains both ATP-dependent and ATP-independent transport systems (Table 1, Fig. 1). ATP-dependent transport
systems in the liver are members of the ATP-binding
cassette (ABC) superfamily, and they transport biliary
constituents against steep concentration gradients across
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Basolateral efflux systems belonging to the multidrug
resistance (MRP/Mrp) subfamily normally are expressed
at only very low levels but may be induced under cholestatic conditions. In humans, this family currently comprises six members (MRP1– 6), at least four of which
(MRP1, -2, -3, -6) have been demonstrated in liver (37,
180). The hepatic expression of MRP4 and MRP5 proteins
is low, and their physiological function and (sub)cellular
localization are not yet known (140). Three members of
this family have also been identified in rat (Mrp2, -3, -6),
five in mouse (Mrp1, -2, -4, -5, -6), and one in rabbit (Mrp2)
(180). The founding member of the MRP family, MRP1
(Mrp1 in rodents), was initially cloned from a multidrug
resistant small lung cancer cell line (73) and is minimally
(if at all) expressed in normal liver (73), where it has been
reported to be localized to the basolateral membrane of
hepatocytes (240). However, potential antibody cross-reactivity with other Mrps should be considered when interpreting these early reports. Mrp3/MRP3 and Mrp6/
MRP6 have also been localized to the basolateral membrane of hepatocytes, while Mrp2/MRP2 is localized to the
canalicular membrane. At the hepatocellular level, rat
Mrp3 is normally only expressed in the terminal cells in
the lobule surrounding the pericentral vein (89, 192, 331).
Mrp3/MRP3 has also been localized to the basolateral
membrane of cholangiocytes (89, 194, 299, 308, 331) and
intestinal epithelia (135, 186, 300, 308), where it may be
involved in the cholehepatic and enterohepatic circulation of bile salts. Under normal conditions, expression of
Mrp3/MRP3 is also very low in hepatocytes in contrast to
cholangiocytes. Human MRP3 is mainly expressed in the
periportal region of the lobule in addition to cholangiocytes (37, 308). Similar to Mrp2/MRP2, Mrp1/MRP1 and
Mrp3/MRP3 are ATP-dependent export pumps whose
spectrum (although with different affinities) include glucuronide and glutathione conjugates of endogenous and
exogenous compounds (180, 191). Mrp1/MRP1 (179, 180)
and Mrp3/MRP3 (6, 137, 138) have been shown to transport divalent bile salts such as sulfated taurolithocholate
and taurochenodeoxycholate with high affinity. In addition, Mrp3 can also transport monovalent bile salts such
as tauro- and glycocholate (6, 137, 138), while human
MRP3 transport only glycocholate with low affinity, but
not taurocholate to a significant degree (5, 398). Mrp1 and
Mrp3/MRP3 are normally expressed at very low levels in
hepatocytes, but are dramatically upregulated in cholestasis in rats (89, 136, 268, 285, 331, 352, 367). Because
Mrp1/MRP1 and Mrp3/MRP3 are able to transport the
divalent anionic bile salts such as sulfated and glucuronidated bile salts that are eliminated into urine in cholestasis, the induction of Mrp1 and Mrp3/MRP3 during
cholestasis may explain the shift toward renal excretion
of bile salts as a major mechanism for bile salt elimination
in patients with chronic, long-standing cholestasis (290,
339). Similarly, Mrp4 RNA has been recently shown to be
upregulated in bile salt-fed mice, suggesting that this
transporter could also be involved in basolateral bile salt
efflux (314). However, further studies will have to demonstrate whether Mrp4 is localized to the basolateral
membrane of hepatocytes. Mrp6 is localized predominantly to the lateral membrane of hepatocytes in rat liver
where it mediates transport of the anionic cyclopentapeptide and endothelin receptor antagonist BQ-123; of note,
other cyclic peptides such as endothelin and vasopressin
are transported by Mrp2 but not by Mrp6 (233). MRP6 is
constitutively and highly expressed in hepatocytes and
kidney (193). Recent evidence suggests that MRP6 may be
involved in transport of glutathione conjugates and leukotrienes (LTC4) (146). Mutations of the MRP6 gene are
associated with pseudoxanthoma elasticum, although the
functional link underlying this observation remains unclear (27, 146, 294).
Members of the Oatp/OATP family also remain candidates for bile salt efflux at the basolateral membrane,
since studies in Xenopus laevis oocytes have shown that
Oatp1 and -2 are able to operate as bidirectional exchangers (228), although this remains to be demonstrated for
hepatocytes.
BILE SALT TRANSPORTERS
Physiol Rev • VOL
of 16-day-old fetuses (400), indicating that biliary excretion of monovalent bile salts does not take place until
birth. Bsep transports various bile salts at rates in the
same order of magnitude as ATP-dependent transport in
canalicular rat liver plasma membrane vesicles. In addition to taurocholate, Bsep also mediates ATP-dependent
transport of glycocholate, taurochenodeoxycholate, and
tauroursodeoxycholate (210, 245). Rat Bsep mediates
low-level resistance to taxol, but not to other drugs (e.g.,
vinblastine, digoxin) that form part of the multidrug resistance phenotype (69). Transfection studies with mouse
Bsep also revealed only slightly enhanced vinblastine efflux, indicating that Bsep is probably not of major relevance for detoxification of xenobiotics (218).
The mouse Bsep gene was localized to a region on
mouse chromosome 2, which has also been linked to
genetic gallstone susceptibility, although the detailed
mechanisms remain unclear (117, 213). In contrast to
humans with a hereditary BSEP defect (PFIC-2), targeted
inactivation of this gene in mice results in nonprogressive
but persistent intrahepatic cholestasis, due to the de novo
formation and biliary excretion of muricholic acid and a
novel tetra-hydroxylated bile salt in mice (376). Of note,
bile flow is minimally reduced and bile salt excretion is
only reduced to 30%, suggesting that additional bile salt
transporter(s) may exist in mice. Although the molecular
identity of this alternative transport system remains to be
determined, Mrp2 is a likely transporter for tetrahydroxy
bile salts (245). Alternatively, it remains possible that
sulfated and glucuronidated dianionic bile salts, which
also are Mrp substrates, account for most of the secreted
bile salts in these animals (180). Mouse Bsep contains
several phosphorylation sites that appear to be involved
in regulating bile salt transport capacity by Bsep (264).
Bsep is highly conserved during vertebrate evolution as
suggested by the recent cloning of a Bsep ortholog from the
liver of the small skate, Raja erinacea, a 200 million-year-old
marine vertebrate (23, 54). Notably, the sites of published
BSEP mutations in humans are also conserved from the
skate Bsep ortholog, and mutations of several of these sites
inhibit skate Bsep transport function in Sf9 cells (54).
Identification of the gene responsible for a subtype of
progressive familial intrahepatic cholestasis (PFIC-2) has
led to the discovery of the human BSEP gene. The human
BSEP gene is mutated in patients with PFIC-2, characterized by absence of BSEP from the canalicular membrane
and extremely low biliary bile salt concentrations that are
⬍1% of normal (see sect. IIIA) (155, 344). This suggests
that BSEP is the major canalicular bile salt transport
system. After several years of effort, the human BSEP has
now been functionally expressed by two groups and appears similar to studies in human canalicular membrane
vesicles with respect to bile salt affinity and substrate
specificity (53, 265).
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the canalicular membrane that are often in the range of
100- to 1,000-fold and are driven by ATP hydrolysis. A
typical ABC transporter consists of 12 or more membrane-spanning domains that determine the substrate
specificity and two intracellular nucleotide-binding loops
that are highly conserved and contain the Walker A and B
motifs for binding and hydrolysis of ATP (140). Most
canalicular ABC proteins belong to the multidrug resistance P-glycoprotein (MDR) family also known as ABC-B
family or the multidrug-resistance protein (MRP) family
(ABC-C) (140, 178). Two other families, ABC-A and ABCG, may also be relevant, since at least three ABC-A members (ABCA1, -2, -3) and three ABC-G half transporters
(ABCG2, -5, and -8) are highly expressed in liver (94, 229).
The canalicular membrane contains a bile salt export
pump (Bsep/BSEP) for monovalent bile salts, a conjugate
export pump (Mrp2/MRP2) for divalent bile salts, and
various other amphipathic conjugates, including GSH (a
major determinant of bile salt-independent canalicular
bile flow), a multidrug export pump (Mdr1/MDR1) for
bulky amphipathic organic cations (e.g., various drugs), a
phospholipid flippase (Mdr2 in rodents/MDR3 in humans)
for phosphatidylcholine, a P-type ATPase (Fic1/FIC1) mutated in hereditary cholestasis but whose function is still
⫺
unknown, and a Cl⫺/HCO⫺
3 exchanger (AE2) for HCO3
excretion. The latter represents the only functionally relevant ATP-independent transport system at the canalicular membrane (238, 243). All other transporters, except
FIC1 (which is a P-type ATPase), belong to the ABC
superfamily. AE2 and MRP2 are the major driving forces
for bile salt-independent bile flow, while BSEP drives bile
salt-dependent flow (205, 258, 259, 342, 359).
A) Canalicular excretion of monovalent bile salts
via Bsep/BSEP. The canalicular bile salt export pump
Bsep/BSEP (Abcb11/ABCB11) originally was known as
the sister of P-glycoprotein (Spgp/SPGP). Following the
partial cloning of Spgp in pig liver (68), the full-length
cDNA was cloned from rat liver (69, 112). Functional
expression of rat Spgp in Sf9 insect cells demonstrated
that Spgp functions as an ATP-dependent bile salt transporter with transport characteristics comparable with
canalicular rat liver plasma membrane vesicles (112).
These functional data, together with hepatocyte-specific
expression of Spgp on the surface of canalicular microvilli, indicate that Spgp represents the mammalian canalicular bile salt export pump. The rat Bsep is a 1,321amino acid protein with 12 putative membrane-spanning
domains, four potential N-linked glycosylation sites, and a
molecular mass of ⬃160 kDa. Bsep was localized by
immunofluorescence microscopy and immunogold labeling studies to canalicular microvilli and to a canalicular
subpopulation of membrane vesicles (112). When ontogenic expression of Bsep was compared with that of
Mrp2, it became evident that Bsep is almost undetectable
before birth, whereas Mrp2 was already observed in livers
641
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MICHAEL TRAUNER AND JAMES L. BOYER
Physiol Rev • VOL
enterocytes, and is mutated in individuals with Tangier
disease and familial HDL deficiency with an increased risk
for atherosclerosis and premature coronary artery disease (34, 48, 301). ABCA1 is highly expressed in liver but
probably does not play a major role in biliary cholesterol
excretion, since Abca1 knock-out mice also do not demonstrate defects in bile salt-induced cholesterol excretion
(A. Groen, personal communication). Mutations of two
highly homologous genes (ABCG5 and ABCG8) that encode the plant sterol sitosterol transporters, sterolin-1
and -2, respectively, have been identified in patients with
sitosterolemia, a disease characterized by impaired biliary
excretion of dietary sterols. ABCG5 and ABCG8 are also
highly expressed in liver (26, 144, 222, 229) and may play
a key role in hepatic cholesterol excretion (222, 273) as
suggested by a fivefold increased biliary cholesterol excretion in mice with transgenic over expression of human
ABCG5 and ABCG8 (397).
Fic1/FIC1 (ATP8B1) a P-type ATPase mutated in “familial intrahepatic cholestasis” belongs to a family of
putative aminophospholipid transporters. Fic1/FIC1 has
been localized to the canalicular membrane and the bile
duct epithelium (361) but is also highly expressed in
extrahepatic tissues including the intestine and pancreas
(50, 96). The detailed function of Fic/FIC1 is unclear, but
mutations of this transporter result in variants of familial
intrahepatic cholestasis, suggesting that it must play an
important direct or indirect role in canalicular bile salt
excretion (50). Of note, these patients have a prominent
reduction in the biliary excretion of hydrophobic bile
salts such as lithocholate and chenodeoxycholate relative
to cholate conjugates, indicating that Fic1/FIC1 could
directly excrete highly hydrophobic bile salts (335). Apart
from this, Fic1/FIC1 could also play an indirect role in bile
secretion by maintaining the canalicular membrane asymmetry between the inner and the outer layer by maintaining phosphatidylethanolamine and serine within the inner
bilayer of the plasma membrane or regulating the docking
of vesicles fusing with the canalicular membrane (276).
Strong expression of Fic1/FIC1 in extrahepatic tissues
such as pancreas, small intestine, and kidney suggests a
more general role in the regulation of secretory processes
(50) and may explain some of the extrahepatic features
associated with these syndromes such as pancreatitis,
diarrhea, and nephrolithiasis. Fic1 (⫺/⫺) mice have normal biliary bile salt excretion but accumulate bile salts
when fed orally due to abnormal regulation of intestinal
bile salt absorption. The detailed mechanisms remain to
be resolved (283).
C. Cholangiocellular Bile Salt Transport
Although contributing only 3–5% to the total liver cell
mass, bile duct epithelial cells (cholangiocytes) play an
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B) Canalicular excretion of divalent bile salts via
Mrp2/MRP2. The bilirubin conjugate export pump (Mrp2/
MRP2) (Abcc2/ABCC2), functionally also known as the
canalicular multispecific organic anion transporter
(cMOAT), was originally cloned from rat liver (49, 280),
followed by human (353) and mouse liver (101). Mrp2/
MRP2 mediates the canalicular excretion of a broad range
of organic anions; most of these are divalent amphipathic
conjugates with glutathione, glucuronate, and sulfate
formed by phase II conjugation in the hepatocyte (e.g.,
bilirubin diglucuronide) (76, 191, 274). Canalicular excretion of GSH is also mediated through Mrp2/MRP2 (279).
Divalent bile salts with two negative charges such as
sulfated tauro- or glycolithocholate are transported by
Mrp2/MRP2, whereas monovalent bile salts are not substrates for Mrp2/MRP2 (179, 180). However, mutation of a
single amino acid confers transport capacity for monovalent bile salts to Mrp2 (149). Other Mrp/MRP isoforms
such as Mrp1/MRP1 and Mrp3/MRP3 are located at the
basolateral membrane where they may serve as a compensatory overflow system under cholestatic conditions
when canalicular (Mrp2/MRP2) excretory function is impaired. The mouse Mrp2 gene was localized to a region on
mouse chromosome 19D2 (214, 363), which has also been
linked to genetic gallstone susceptibility (213). The human MRP2 gene has been mapped to chromosome 10q24
(363). Mutations of the MRP2 gene result in the DubinJohnson syndrome characterized by impaired canalicular
excretion of a broad range of endogenous and exogenous
amphipathic compounds (see sect. IIIA); the rat model for
this syndrome is the transport deficient (TR⫺) and Groningen Yellow (GY) rat (Wistar strain) or Eisai hyperbilirubinemic (EHBR) rat (Sprague-Dawley strain), which
were pivotal models for the functional characterization of
cMOAT and subsequent cloning of Mrp2 (281).
C) Other canalicular transport systems involved in
bile secretion and their relation to bile salt excretion.
Once bile salts have been excreted into bile, they stimulate the release of phosphatidylcholine and cholesterol
from the outer leaflet of the canalicular membrane (272),
which then in turn form mixed micelles in bile. By doing
so, bile salt toxicity to the bile duct epithelium is avoided,
which would otherwise occur due to an unopposed detergent bile salt action. The phospholipid flippase Mdr2/
MDR3 (Abcb4/ABCB4) ensures the continuous supply of
phosphatidylcholine to the outer leaflet of the canalicular
membrane (327, 362).
In addition to biliary excretion of phospholipids, bile
is also a major pathway for elimination of cholesterol,
which is mostly derived from plasma high-density lipoprotein (HDL). Hepatocellular uptake of HDL cholesterol is
mediated via a scavenger receptor (SR-BI) (3, 158, 195).
An ATP-dependent cholesterol transporter (ABCA1) mediates reverse cholesterol transport from macrophages
into HDL particles, removes absorbed cholesterol from
BILE SALT TRANSPORTERS
important role in normal bile secretion (39, 355). Large
and medium-sized but not small (⬍30 ␮m in diameter)
intrahepatic bile ducts contain several transport systems
for secretory and absorptive functions (168) (Table 1, Fig.
1). Biliary constituents including bile salts, glucose, and
drugs may be transported from bile into cholangiocytes
However, only a minority of bile salts are in solution in
bile as free monomers at the level of the bile ducts, and
thus only small amounts of bile salts might be available
for absorption by cholangiocytes (245).
1. Apical transport systems in biliary epithelia
2. Basolateral efflux from cholangiocytes
After uptake, bile salts are effluxed via the basolateral membrane of cholangiocytes into the peribiliary
plexus. Functionally, this mechanism has been characterized as Na⫹ independent (25). At a molecular level, this
may be achieved by Mrp3/MR3, which has been identified at
the basolateral membrane of cholangiocytes and gallbladder
epithelium in rats and humans (89, 194, 299, 308, 331).
Additional transport systems may involve an alternative (truncated) splicing variant of Isbt/Asbt (t-Asbt) for
which no driving force has as yet been established (217).
Expression studies in oocytes predict that t-Asbt in contrast to Asbt acts as an efflux carrier (217). The latter
observation would suggest that two Asbt isoforms are
required for cholangiocellular bile salt transport to occur,
although the existence of t-Asbt is being questioned recently. Alternatively, Oatp3 could be involved in basolateral bile salt efflux from cholangiocytes (210).
Physiol Rev • VOL
D. Intestinal Bile Salt Transport
Bile salts, cholesterol, and phospholipids undergo
extensive enterohepatic cycling/enterohepatic circulation, thereby returning these biliary constituents to the
liver for reexcretion into bile (139, 156, 245) (Table 1, Fig.
1). The most efficient bile salt conservation mechanism is
the uptake of conjugated bile salts in the terminal ileum
via a Na⫹-dependent mechanism (74, 342). In addition, a
Na⫹-independent anion exchanger has been identified in
proximal rat jejunum (12). Passive diffusion of unconjugated bile salts also occurs in small and large intestine
(246).
1. Apical uptake of bile salts into enterocytes
Na⫹-dependent bile salt uptake occurs via the ileal
bile salt transporter (Isbt/ISBT or Ibat/IBAT) also known
as the apical sodium-dependent bile salt transporter
(Asbt/ASBT). This transport system has also been identified in the apical membrane of cholangiocytes and proximal renal tubular cells. Isbt was originally cloned from
hamster intestine (387), followed by human (388), rat
(320), rabbit (198), and mouse (302) ileum. Size (48 kDa),
membrane topology, and transport characteristics are
similar to Ntcp/NTCP (35% amino acid identity) (79, 129,
342). Isbt is expressed biphasically during rat development, with the first expression on day 22 of gestation,
followed by a transient decrease and a sharp increase at
postnatal days 17 and 18 (320, 322). Both primary and
secondary conjugated and unconjugated bile salts are
substrates for Isbt/ISBT, the highest affinity being reported for conjugated dihydroxy bile salts (267). However, in contrast to Ntcp/NTCP, which transports some
nonbile salt substrate in addition to bile salts, the substrate specificity of Isbt/ISBT appears to be strictly limited
to bile salts. ISBT is the major intestinal bile salt uptake
system in humans as emphasized by ISBT mutations that
result in bile salt malabsorption (267, 388).
The Na⫹-independent bile salt transporter Oatp3
(Slc21a7) is 80 – 82% identical to Oatp1 and Oatp2 and has
transport characteristics similar to Oatp2, with a range of
amphipathic anions as its substrates, including bile salts
(59, 370). Oatp3 was originally cloned from retina and was
found to be expressed in the brush-border membrane of
jejunal enterocytes (1, 370). Oatp3 mRNA transcripts
were detected throughout the entire small intestine (as
well as brain, lung, and kidney), but Oatp3 protein is
predominantly located to the apical surface of jejunal
epithelial cells (1, 370), consistent with a role of Oatp3 as
the Na⫹-independent transport system which has been
functionally localized to the jejunum. Bile salt uptake into
jejunal brush-border membrane vesicles is stimulated by
an in-to-out HCO⫺
3 gradient, similar to Oatp1 (303). However, it remains to be determined whether this mechanism
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Bile salts are taken up by Isbt/ISBT, also called Asbt/
ASBT (SLC10A2) (216), which is identical to the gene
product expressed in the terminal ileum. However, the
relative abundance of Isbt in rat cholangiocytes is about
sevenfold lower than in the ileum (8, 216), possibly indicating different physiological roles such as signaling
through bile salts. A minor part of reabsorbed bile salts
may also be taken up in their protonated form by nonionic
diffusion (139).
The gallbladder epithelium has considerable capacity
to modify the composition of primary hepatic bile by
absorptive and secretory processes. MRP2 and MRP3
were recently identified in human gallbladder epithelia.
MRP2 was located in the apical membrane and MRP3 in
the basolateral membrane as in other epithelia. Of note,
MRP1 protein expression was not detectable (299). Moreover, ISBT and OATP-A expression were detected in a
human gallbladder-derived biliary epithelial cells where
these transporters may mediate sodium-dependent and
-independent taurocholate uptake (67).
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MICHAEL TRAUNER AND JAMES L. BOYER
also applies to Oatp3. The relative importance of Oatp3
for intestinal bile salt uptake compared with Isbt also
remains to be clarified (342). Interestingly, OATP-A has been
proposed as the human Oatp3 ortholog (370), and human
OATP-A has been detected in human intestine (245).
In addition to uptake systems in the apical membrane, the apical brush-border membrane also contains
excretory systems, including members of the Mdr and
Mrp family (e.g., Mdr1, Mrp2) (Fig. 1). As far as bile salt
transport is concerned, Mrp2 could play a role as an
alternative excretory route for sulfated/glucuronidated
bile salts (in analogy to the role of Mrp2 in kidney, see
sect. IE).
As with other cells involved in bile salt transport, the
information about the molecular mechanisms of intracellular bile salt transport in enterocytes is limited. Photoaffinity labeling studies have identified a 14-kDa cytosolic
intestinal bile acid-binding protein (I-Babp) that is cytoplasmatically attached to Isbt (115, 196). I-Babp probably
represents the most important protein for transcellular
bile salt movement through enterocytes (197). The functional ileal bile salt uptake complex is multimeric and
comprises four Isbt dimers and four I-Babps (196). Similar
ontogenic expression patterns of Isbt and I-Babp as well
as their response to bile salts and dexamethasone suggest
that both transport systems might be controlled by similar
if not identical regulatory mechanisms (145, 320).
3. Basolateral efflux of bile salts from enterocytes
At a functional level, an anion exchange mechanism
has been demonstrated at the basolateral membrane of
intestinal cells (381). Recently t-Asbt (which can function
as an anion exchanger) has been reported to be expressed
twofold higher at the mRNA level than the full-length Asbt
in rat ileum (217). Another potential candidate for bile salt
efflux from enterocytes is Mrp3/MRP3, which has also
been identified in both rat and human small intestine (135,
186, 300, 308). Mrp3/MRP3 is expressed in the basolateral
membrane in all intestinal segments but is lowest in duodenum and markedly increased in the terminal ileum and
colon (330). This is in contrast to Mrp2/MRP2, which is
expressed primarily in the apical membrane of the proximal
intestine (65, 300, 348). The high expression of Mrp3/MRP3
in terminal ileum provides a mechanism to return bile salts
to the portal circulation. Mrp3/MRP3 may also play an important role in drug absorption in the intestine.
E. Renal Bile Salt Transport
Bile salts that escape first-pass clearance by the liver
are filtered at the glomerulus from plasma into urine, where
Physiol Rev • VOL
1. Apical transport systems in proximal renal tubules
The apical plasma membrane of the proximal renal
tubular cells contains transport systems for bile salt reabsorption as well as excretion into urine (342). Bile salts
are reabsorbed by the apical Na⫹-dependent bile salt
transporter Isbt in the proximal convoluted tubule (71). In
addition to Isbt, Mrp2 (306) and Oatp1 (28) have also been
localized to the apical brush-border membrane. Mrp2 may
be involved in tubular excretion of organic anions (e.g.,
para-aminohippuric acid) under normal conditions and
increased urinary excretion of sulfated/glucuronidated
bile salts under cholestatic conditions (306). In contrast
to its basolateral localization in hepatocytes, Oatp1 is
localized to the apical membrane of the S3 segment of
proximal renal tubules (28). The relative contribution of
Oatp1 to tubular bile salt uptake remains unclear. Moreover, Oatp3 (Slc21a7) has also been reported to be expressed in rat kidney (1), although this remains controversial (342). By analogy to the rat, ISBT (74), OATP-A
(207), and MRP2 (305) have also been localized in human
proximal kidney tubular cells.
2. Basolateral efflux systems in proximal
renal tubules
Little is known about the basolateral counterparts of
the apical transport systems described above. Recent evidence suggests a potential role for Mrp1 (285). However,
Mrp3 has recently been localized to the basolateral membrane of the proximal renal tubule in rats (330). Therefore, it will also be of particular interest to see whether
Mrp3 also plays an important role for basolateral efflux,
e.g., of bile salts.
F. Placental Bile Salt Transport
Because the fetal liver is immature and ontogenetic
expression of hepatobiliary transporters is not detectable
until shortly before birth (19), bile salts undergo minimal
biliary excretion by the fetal liver, although bile salts are
synthesized by the fetal liver in utero (260). Instead, they
are eliminated by the maternal liver after vectorial translocation from the fetal to the maternal circulation via the
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2. Intracellular bile salt transport in enterocytes
they are reabsorbed in the proximal convoluted tubule (386)
(Table 1, Fig. 1). Thus, under normal conditions, urinary bile
salt losses are minimized. Under cholestatic conditions however, renal excretion of bile salts may become a major
alternative elimination route for elimination of (mainly divalent sulfated and glucuronidated) bile salts (290), which may
be attributed to increased passive glomerular filtration of
(elevated) serum bile salts and active tubular excretion of
(mainly divalent) bile salts, together with reduced tubular
bile salt conservation.
645
BILE SALT TRANSPORTERS
placenta (“placenta-maternal liver tandem”). The bloodplacental barrier of term placenta begins at the fetal side
with the endothelial cells of fetal capillaries, cytotrophoblast (absent in term placenta), and syncythiotrophoblasts that face the maternal blood with their apical surface. Distinct transport systems have been identified at a
functional level at the basolateral (fetal-facing) and apical
(maternal-facing) membrane of the trophoblast. However,
the molecular identity of these transport systems has not yet
been resolved, and data are still quite fragmentary (342).
1. Basolateral (fetal-facing) trophoblast membrane
2. Apical (maternal-facing) trophoblast membrane
Both ATP-dependent and ATP-independent bile salt
transport systems have been identified, the latter appearing
to be the predominant transport mechanism (46, 342). However, BSEP remains a candidate, since partial Bsep/BSEP
transcripts have been identified in term placenta (342).
II. REGULATION OF BILE SALT
TRANSPORTERS IN NORMAL PHYSIOLOGY
The functional expression of membrane transport
proteins can be regulated at several levels, including gene
transcription, events that control translation of RNA, and
posttranscriptional activity (see Table 2 and Figs. 2 and
3). Although the mechanisms that control gene transcription of membrane transporters are still incompletely un-
TABLE 2. Nuclear hormone receptors and other transcription factors that regulate bile salt
metabolism enzymes and transporters
Nuclear Hormone Receptors
and Other Transcription
Factors
Activating Ligand for the Nuclear
Receptor
LXR (liver X receptor)
Oxysterols
SHP-1 (short heterodimeric
protein-1)
RAR (retinoic acid
receptor)
FXR (farnesoid X activated
receptor)
PXR (pregnane X receptor
in rodents), SXR (steroid
X receptor in humans)
None
CAR (constitutive
androstene receptor)
NF␬B (nuclear factor ␬B)
HNF-3␤ (hepatic nuclear
factor-3␤)
HNF-4␣
All trans-retinoic acid
Hydrophobic bile salts
Xenobiotics; ursodeoxycholate,
lithocholate; pregnenolone
16␣-carbonitrile (PCN) in
rodents); rifampin (in
humans)
Xenobiotics
Cytokines
Upregulates bile salt synthesis from
cholesterol via CYP7A1
Downregulates bile salt uptake via Ntcp and
bile salt synthesis via CYP7A1
Upregulates Ntcp and Mrp2
SHP-1, Bsep/BSEP, I-BABP,
Mrp2, OATP8
Oatp2 in rodents; CYP3A4,
Mrp2
Upregulates SHP-1; Bsep/BSEP I-BABP and
OATP8
Upregulates Oatp2 and Mrp2; upregulates
CYP3A and stimulates detoxifying bile salt
hydroxylation
CYP3A, Mrp2
Stimulates hepatic drug metabolism, and drug
conjugate export pump, Mrp2
Stimulates upregulation of Mdr1b
Downregulates these transporters when
overexpressed in mice
Maintains Ntcp and Oatp1 expression;
upregulates Bsep and downregulates Mdr2
in knock-out mouse
Loss of expression of Ntcp, Oatp1, Oatp2, and
Isbt in knock-out mouse
Ntcp, Oatp1, Bsep, Mdr2
Ntcp, Oatp1, Oatp2, Oatp-4,
Isbt; human OATP-C,
OATP-8
Isbt
MRP3 CYP8B1
? Bile salts
? Bile salts
Fibrates, fatty acids,
eicosanoids, leukotrienes,
NSAIDS
Physiol Rev • VOL
Expected Physiological Effect
CYP7A1 (cholesterol 7-␣
hydroxlase)
CYP7A1 (cholesterol 7-␣
hydroxylase), Ntcp
Ntcp, Mrp2
MDR1
Ntcp, Mdr2
HNF-1␣
AP-1 (activating protein-1)
FTF (fetal transcription
factor)
PPAR␣ (peroxisome
proliferator activator-␣)
Regulation of Bile Salt
Metabolism Enzymes and
Transporter Genes
ISBT
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Upregulates Isbt in vitro
Upregulates MRP3 in vitro; downregulates
CYP8B1 in vitro
Upregulates ISBT in vitro
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Bile salt uptake occurs via Na⫹-independent, bidirectional, and HCO⫺
3 trans-stimulatable mechanisms, consistent with a role for OATPs/Oatps. Notably, partial OATP-A
(SLC21A3) transcripts have been identified in term placenta (342). Recently, OATP-B (SLC21A9) was localized
to the basal syncythiotrophoblast and cytotrophoblast
membranes, where it may be involved in the placental
uptake of fetal-derived sulfated steroids (341). Preliminary data indicate the presence of Oatps 1, 2, and 4 mRNA
transcripts in rat placenta (J. J. Marin, personal communication). Moreover, recent studies have localized MRP3
in fetal blood endothelia of term placenta and syncytiotro-
phoblast layer, suggesting a potential role for MRP3 in the
extrusion of fetal bile salts through endothelial and syncythiotrophoblast barriers (343). Similarly Mrp1, -2, and -3
transcripts have recently been detected in rat placenta,
although their subcellular localization remains to be determined (Marin, personal communication).
646
MICHAEL TRAUNER AND JAMES L. BOYER
availability of RXR, limiting cellular/nuclear RXR concentrations may result in a trans-repressive effect.
In addition to these ligand-activated nuclear receptors, other factors such as the hepatocyte nuclear factor
(HNF) family of liver-enriched transcription factors including HNF-1 (318, 319), HNF-3 and CCAAT/enhancer
binding protein (C/EBP), as well as sterol responsive
element binding protein (SREBP) and nuclear factor
kappa B (NF-␬B) also appear to play an important role in
the regulation of hepatobiliary transporter expression (60,
257, 360) (Table 2).
A. Transcriptional Regulation of Bile Salt
Transporters
1. Role of nuclear hormone receptors in regulation
of bile salt transporters
Table 2 and Figure 2 list several of the bile salt
transporters whose expression appears to be regulated by
one or more NHRs. Bile acids are ligands for the nuclear
hormone receptor FXR, which together with its heterodimeric partner, the RXR, acts as a transcription factor
for several bile salt transporters, including the hepatic
bile salt export pump Bsep and the ileal bile acid binding
protein I-Babp (235, 277, 373). In addition, the expression
of short heterodimeric protein (SHP)-1, which acts as a
transcriptional repressor is itself regulated by FXR and
can downregulate the expression of several genes including Ntcp and cholesterol-7a-hydroxylase CYP7A1, the rate
limiting enzyme in bile salt synthesis. Recent studies suggest that the bile salt lithocholate and its 6-hydroxylated
metabolite are also ligands for the PXR, resulting in the
FIG. 2. Role of ligand-activated transcription factors in the transcriptional
regulation of bile salt synthesis and
transport. Conversion of cholesterol to
bile salts by CYP7A1 and 8B1 is stimulated by oxysterol-activated liver X receptor (LXR). Bile salts inhibit their own
synthesis by farnesoid X receptor (FXR)dependent activation of SHP-1, which in
turn suppresses CYP7A1 and 8B1 transcription. Bile salts also suppress retinoic acid receptor (RAR)-dependent
genes such as Ntcp via the same pathway. Activation of FXR by bile salts stimulates transcription of Bsep, Mrp2,
I-Babp, and OATP8. Activation of pregnane X receptor (PXR) by bile salts induces transription of Oatp2 and Mrp2.
Finally, liver receptor homolog-1 (LRH-1)
may mediate induction of MRP3 by bile
salts in human enterocytes. RXR, retinoid
X receptor; FTF, fetoprotein transcription
factor.
Physiol Rev • VOL
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derstood, a number of factors may be involved including
constitutive and inducible DNA binding proteins that bind
to upstream regulatory elements (RE) in the gene promoters. These transcription factors often act together to either stimulate or inhibit gene expression (17). Recent
interest has focused on a group of nuclear hormone receptors (NHR) that are activated by various ligands such
as steroids, retinoids, and hormones (61, 62). The nuclear
receptor superfamily is divided into four major subgroups
based on their dimerization and DNA binding properties
(for reviews, see Refs. 66, 169, 236, 237). Class I contains
the classical steroid hormone receptors such as for estradiol, progesterone, testosterone, cortisol, and aldosterone. Class II receptors comprise the receptors for vitamin D3 (which recently has been shown to be also activated by the hydrophobic bile salt lithocholate, Ref. 234),
T3, and all-trans-retinoic acid. The latter (all-trans-retinoic
acid receptor RAR) is also involved in the regulation of
hepatobiliary transporter genes as outlined below. Other
class II receptors involved in the regulation of bile salt
synthesis and transport include the farnesoid X receptor
(FXR) that responds to bile salts; peroxisome proliferatoractivated receptor (PPAR␣), whose high-affinity ligands are
fatty acids, eicosanoids, fibrates, and NSAIDS; liver X receptor (LXR) that binds oxysterols; and the pregnane X receptor
(PXR) whose human ortholog is the steroid and xenobiotic
receptor (SXR) (392). PXR and SXR receptors are the targets for catatoxic steroids and certain xenobiotics. Class II
receptors heterodimerize with the retinoid X receptor (RXR)
enabling high-affinity DNA binding to direct repeats (DR)
separated by spacer nucleotides of variable number (DR1–
5), which then leads to activation of gene transcription.
Because heterodimer formation largely depends on the
BILE SALT TRANSPORTERS
Physiol Rev • VOL
human ileal bile acid binding protein (I-BABP) by bile
salts was dependent on FXR/RXR binding to the highly
conserved IR-1 (G/A GGTA A TAACCT) (118, 165, 166,
235).
FXR is expressed in tissues where bile salts are transported including the liver, intestine, and kidney and is
activated by bile salts with the rank order of potency
chenodeoxycholate (CDCA) ⬎ deoxycholate (DCA) ⫽
lithocholate (LCA) ⬎ cholate (CA) (277). Evidence that
FXR is the nuclear bile acid receptor (BAR) came from
studies where expression plasmids containing murine and
human FXR were transfected into monkey kidney CV-1
cells or human hepatoma HepG2 cells and exposed to bile
salt metabolites (235). In these experiments CDCA was
the most potent activator, with a half-maximal effective
concentration (EC50) of 50 and 10 ␮M for murine and
human FXR, respectively. Amino acid residues asparagines (354) and isoleucine (372) confer sensitivity of human FXR to CDCA (75). DCA and LCA also stimulated
FXR expression but to a lesser extent, whereas other
sterols including cholesterol, oxysteols, steroid hormones, and other bile acid metabolites had no effect (235,
277). Particularly noteworthy was the absence of an activating effect by ursodeoxycholate (UDCA), a commonly
used bile acid for the treatment of cholestasis (235), and
␤-muricholic acid, a prominent bile acid that accumulates
in the cholestatic rat model (316). Cotransfection assays
indicated that FXR/RXR heterodimers were required for
bile acids to maximally transactivate the luciferase reporter. In the same studies, FXR was found to repress
transcription of the gene encoding for cholesterol 7␣hydroxylase (CYP7A1), the rate-limiting enzyme in bile
acid synthesis from cholesterol while stimulating the expression of the human intestinal bile acid binding protein
(I-BABP) (235), a 17-kDa protein with high affinity for bile
acids, that modulates bile salt uptake in the terminal
ileum. Studies in Caco-2 cell lines, derived from human
enterocytes, show that bile salts increase I-BABP expression, a finding consistent with the effects of bile diversion
and cholystyramine administration in mice where I-Babp
expression is diminished (118). Deletion and mutation
analyses have established that the FXR/RXR␣ heterodimer activates I-BABP gene expression by binding to
bile acid response elements in its promoter as demonstrated in human, mouse, and rabbit (118). Subsequent
studies have demonstrated that FXR activates the expression of the transcriptional repressor SHP-1, which functions in a dominant manner to inhibit CYP7A1 in the
mouse (see below). Thus bile salts feedback to regulate
their own synthesis by binding to FXR and subsequent
activation of SHP (Fig. 2). Subsequent studies confirm
that both human, rat, and mouse BSEP/Bsep promoters
are transcriptionally activated by FXR (13, 110, 287) and
that bile salts increase BSEP expression in primary human hepatocytes or HepG2 cells with the same rank order
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transcription of CYP3A, an enzyme involved in hydroxylation and detoxification of bile salts (334, 393). Two
␣1-fetoprotein transcription factor (FTF) also known as
liver receptor homolog-1 (LRH-1)-like elements have also
been described in the promoter region of MRP3 that
function as bile salt response elements (147). Thus a
picture is emerging where bile salts may regulate the
expression of several important bile salt-transporting and
-metabolizing systems by binding to various nuclear receptors (Fig. 2).
A) RXR. RXR is the obligate heterodimeric partner
for the class II nuclear receptors and is activated by
binding of 9-cis-retinoic acid. Thus alterations in the expression of RXR would be capable of influencing the
expression of a large number of bile salt synthesis and
transporter genes. RXR␣ is highly expressed in liver, kidney, muscle, lung, and spleen, whereas two other RXR
isoforms, RXR-␤ and -␥, are not as highly expressed (371).
Although RXR-␣ knock-out mice do not survive beyond midgestation, the RXR-␣ gene has been deleted in
adult animals specifically in the liver using cre-mediated
recombination under the influence of an albumin promoter (371). These studies demonstrate that multiple metabolic pathways in the liver that are regulated by RXR
heterodimerization are markedly altered when RXR-␣ is
absent. These include FXR, which functions with RXR to
enhance SHP expression, thereby suppressing the conversion of cholesterol to bile salts. The absence of RXR led to
a significant increase in CYP7A1 mRNA (371). RXR/FXR
appears to have a dominant negative influence on CYP7A1
expression, since RXR/LXR normally promotes the expression of CYP7A1 to stimulate conversion of cholesterol to bile salts. Other class II nuclear receptors that
influence hepatic metabolic pathways are also impaired in
the absence of RXR-␣ including RXR/CAR-␤ and RXR/
PXR (371), leading to marked reductions in mRNA levels
for liver fatty acid binding proteins and CYP4A1 and
CYP2B10 and CYP3A1.
Thus the expression of RXR is one mechanism
through which a coordinated effect on multiple gene
products that influence bile salts, fatty acid, cholesterol,
steroid, and xenobiotic metabolism, might be coordinated.
B) RAR. RAR-␣ together with its heterodimer partner
RXR activate the hepatobiliary transporters Ntcp and
Mrp2 by binding to retinoid response elements in the
promoter regions of these genes. The liver is the major
storage site for vitamin A. Retinoids stimulate expression
and activity of RXR:RAR-dependent genes such as Ntcp
and Mrp2 (81, 345).
C) FXR/bile acid receptors. FXR functions as a heterodimer with RXR and binds with high affinity to inverted repeat response elements (IR-1) where consensus
receptor binding hexamers are separated by a single nucleotide. Initial studies demonstrated that induction of the
647
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MICHAEL TRAUNER AND JAMES L. BOYER
Physiol Rev • VOL
binding sites in the promoter regions of all three genes
(116). When HepG2 cells were transfected with FXR expression vectors controlled by a rat or human SHP-1
promoter, a potent synthetic nonsteroidal ligand
(GW4064) increased expression of a luciferase reporter
gene significantly, indicating that SHP-1 expression is directly regulated by FXR/RXR in these species (116). Bile
acid feeding markedly upregulates the expression of
SHP-1 mRNA while reducing the expression of CYP7A1 in
the mouse (230). Thus bile salts are able to regulate their
own synthesis from cholesterol by stimulating the expression of SHP-1, which in turn inhibits the expression of
CYP7A1 by blocking the function of LRH-1. Bile salt synthesis may also be regulated by sterol 12␣-hyroxylase
(CYP8B1), an enzyme whose gene also contains an LRH-1
response element which may be targeted by SHP-mediated suppression (128). Thus the synthesis of bile salts is
exquisitely regulated by the complex interaction of at
least five nuclear receptors including RXR, LXR, FXR,
SHP-1, and LRH-1 (Fig. 2). However, partial maintenance
of negative feedback regulation of bile salt synthesis in
SHP null mice suggests the existence of SHP-independent
pathways (181, 374), indicating that redundant mechanisms regulate this critical step in bile salt homeostasis
(311). Because bile salt feeding downregulates Ntcp expression in the mouse (326), it is likely that this downregulation results from SHP-1 inhibiting the expression of
this bile salt transporter. Indeed, recent evidence suggests
that inhibition of Ntcp promoter activity by cholic acid in
vitro is mediated through bile salt-induced expression of
the FXR target gene SHP-1 and subsequent direct inhibition of RXR, resulting in inhibition of retinoid transactivation of Ntcp by bile salts (85). These findings provide an
explanation for downregulation of Ntcp expression in
cholestasis (see sect. III). To add to the complexity, SHP-1
can also inhibit transactivation of HNF-4 and RXR (223).
Finally, ligands that activate or inhibit SHP-1 expression
would be candidates for pharmacological manipulation of
bile salt synthesis and of potential benefit in cholestatic
liver disease.
Studies in primary rat hepatocytes indicate that taurocholate as well as tumor necrosis factor (TNF)-␣ are
strong activators of c-Jun NH2-terminal kinase (JNK) and
that SHP-1 is a direct target of activated c-Jun (122).
Mutations in a putative activated protein-1 (AP-1) element
in SHP-1 suppresses c-Jun-mediated activation of the
SHP-1 promoter, indicating that bile acid activation of the
JNK signaling cascade is important in regulating CYPA1
levels in rat hepatocytes (122). Hydrophobic bile salts are
also potent inducers of cytokine expression (TNF-␣ and
interleukin-1) in macrophages (252), indicating that bile
salt-induced cytokines themselves could mediate part of
the bile salt effects. However, these effects appear to be
restricted to unconjugated bile salts. Importantly, both
bile salts and cytokines are known activators of the JNK
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of potency that activates FXR (314). Conversely, LCA
decreases BSEP expression by antagonizing FXR activation (396). Recent studies have demonstrated transactivation of Mrp2 (173) and the human OATP8 promoter by
FXR (162). In contrast to hepatic bile salt transporters
and I-BABP, the ileal sodium-dependent bile salt transporter Isbt was not regulated by FXR, although the studies confirmed a role for FXR in regulation of I-Babp.
In summary, the findings that FXR is abundantly
expressed in tissues that express bile salt transporters,
that CDCA, DCA, and LCA and their conjugates bind to
FXR at physiological concentrations that regulate gene
transcription and are highly effective activators of FXR
when expressed in cellular expression systems, provide
strong evidence that bile salts are the natural ligand for
this NHR.
Confirmatory evidence for the importance of FXR in
the regulation of expression of several bile salt transporters came from studies with targeted disruption of the
nuclear receptor FXR (326). When a 1% cholic acid diet
was fed to wild-type mice, no ill effects were observed.
Ntcp and CYP7A1 were both downregulated while Bsep
was upregulated, thereby limiting the toxic accumulations
of bile salts in the liver. In addition, SHP was also upregulated, facilitating downregulation of Ntcp and reducing
expression of CYP7A1. However, when the FXR null mice
were fed cholic acid, no changes in the expression of
Ntcp, CYP7A1, Bsep, or SHP were observed and the mice
developed severe hepatic necrosis, wasting, and 30% died
by 7 days (326). Altogether these findings emphasize the
importance of FXR in the regulation of hepatic bile salt
transport and support prior studies that suggested that
the downregulation of Ntcp and upregulation of Bsep in
models of cholestasis might function to protect the liver
from accumulation of hepatotoxic levels of bile salts (220)
(Fig. 2).
D) SHP-1. Bile salts are known to repress the expression of CYP7A1, thereby providing feedback regulation
for conversion of cholesterol to bile salts. Conversely,
cholesterol stimulates forward regulation of CYP7A1, the
rate-limiting enzyme for bile salt synthesis from cholesterol (231). The nuclear receptor LXR-␣ is known to be
activated by the oxysterol cholesterol derivative 24,25(S)epoxycholesterol and binds together with its heterodimeric partner RXR to an LXR response element in
the CYP7A1 promoter, thereby inducing CYP7A1 expression. However, the CYP7A1 promoter does not contain
response elements for FXR. In contrast, the nuclear receptor SHP-1, which lacks a DNA binding domain, is
capable of repressing CYP7A1 expression by binding and
repressing the transcriptional activity of liver receptor
homolog (LRH)-1, another monomeric (116, 230) orphan
nuclear receptor that acts as a competence factor and is
required for constitutive CYP7A1 expression (Fig. 2).
SHP-1 from mice, rats, and humans contains RXR/FXR
BILE SALT TRANSPORTERS
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SXR and inducer of CYP3A, has been shown to be effective in the treatment of the symptoms of pruritus in cholestatic liver diseases (21, 56). It has been speculated that
rifampicin may stimulate 6␣-hydroxylation of bile acids,
leading to glucuronidation by UDP-glucuronosyl transferases and excretion of bile salts by previously described
alternative pathways in the urine (18, 47, 289, 385). This
may also explain why cholestasis also improves in some
patients treated with rifampicin (56). Studies in FXR/BAR
nullizygous mice, where the expression of the bile salt
export pump is diminished and hepatic levels of bile salts
increase, report strong upregulation of CYP3A11 and
CYP2B10. Mrp3 and particularly Mrp4 mRNA levels were
also upregulated and enhanced by bile salt feeding in FXR
knock-out mice (314). These studies support the notion
that there are compensatory changes in hepatic transporters that mediate alternative pathways for bile salt excretion that are mediated by bile salts via nuclear receptors.
Interestingly, the hydrophilic bile salts UDCA and its taurine conjugate TUCDA, which are weak activators of FXR,
are potent stimulators of PXR and CYP3A4 in human
hepatocytes, suggesting a possible mechanism for beneficial effects of UDCA in chronic cholestatic liver diseases
(314).
SXR also activates MDR1 gene expression, mediating
the effects of drugs (e.g., paclitaxel, Taxol) on the expression of P-glycoprotein (350). Other studies suggest that
PXR is also involved in regulation of Oatp2 (334), explaining how microsomal-inducing chemicals upregulate
Oatp2 expression (121, 128). Further evidence that PXR
might regulate Mrp2 transcription is provided by studies
on the effects of herbicides in mice in vivo (384) and
xenobiotics (e.g., vincristine, tamoxifen, or the “classic”
PXR-ligand rifampicin) in isolated rat hepatocytes in vitro
(174). On the basis of these observations, it is likely that
SXR/PXR mediate the regulation of Mrp2 and Mrp3 by
drugs and various toxins (e.g., cisplatin, rifamycin) (312).
These findings also suggest that there is a highly coordinated regulation of hepatic endo-/xenobiotic (phase 1)
metabolism (via Cyps) and excretion of their (phase 11)
conjugated metabolites (via Mrp2). Thus a picture is beginning to emerge where SXR/PXR coordinately regulates
uptake, metabolism, and efflux of drugs and potential
toxins including bile salts when they accumulate in the
liver.
G) Constitutive androstane receptor. Like FXR and
PXR, constitutive androstane receptor (CAR) binds DNA
as a heterodimer with RXR␣ but is highly and constitutively expressed in the liver. CAR is inhibited by androstane metabolites (for review, see Ref. 377). In contrast to
other class II NHRs, CAR is also located in the cytoplasm
and translocates to the nucleus after exposure to a diverse group of compounds including phenobarbital,
which is the prototype (380). Recent studies indicate that
CAR and PXR bind to common response elements in the
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pathway resulting in the formation of AP-1 (122, 134). Of
note, an AP-1 element has also been identified in the rat
Isbt promoter, which could mediate bile salt effects on
Isbt expression (333) (Fig. 2).
E) FTF (also known as LRH-1 or CYP7A1 promoter
binding factor). A putative bile salt response element has
been identified in the promoter region of human MRP3
isolated from Caco-2 cells. In this study, CDCA stimulated
MRP3 mRNA threefold in a dose- and time-dependent
manner. Bile salt response elements were identified in the
region ⫺229/⫺138 and included two FTF-like elements.
Since MRP3 is located on the basolateral membrane of
enterocytes, these findings suggest that bile salts may
regulate their enterohepatic circulation from the intestine
(147). Moreover, this mechanism could possibly also explain upregulation of Mrp3/MRP3 in hepatocytes and
cholangiocytes under cholestatic conditions with bile salt
retention. Preliminary studies suggest that an LRH-1 response element may regulate Isbt expression (63) and
could mediate bile salt effects. Conversely, another study
suggests that bile acid-induced FTF may suppress
CYP8B1 expression, possibly by interfering with constitutive transactivation via HNF4␣, which maintains basal
CYP8B1 transcription (394).
F) SXR/PXR. The human SXR and its rodent ortholog
PXR function as xenobiotic receptors that regulate the
expression of CYP3A genes involved in detoxification
pathways for many drugs (223, 390, 391). These cytochrome P-450 enzymes catalyze hydroxylation reactions
that are usually the first step in drug detoxification pathways. Targeted disruption of the mouse PXR gene abolishes the ability of xenobiotics to induce CYP3A. Recent
studies suggest that bile salts may also serve as functional
ligands for SXR and PXR, thereby upregulating CYP3A
enzymes (334, 393) and possibly transporters for hepatic
uptake of drugs (e.g., Oatp2) (121) (Fig. 2). In contrast to
FXR, the rank order of ligand potency is different: 3-ketoLCA ⬎ LCA ⬎ DCA ⫽ CA (334, 393). LCA is a potent
cholestatic bile salt that produces severe hepatic necrosis
when fed to mice. Treatment of mice with pregnolone-16␣-carbonitrile (PCN), a potent inducer of CYP3A, enhances the formation of hydroxylated metabolites of
lithocholic acid and protects mice from the toxic effects
of this bile salt (334, 393). This protective effect of PCN
was abolished in PXR (⫺/⫺) mice (334, 393). Feeding LCA
to PXR (⫺/⫺) mice also failed to change the expression of
FXR and SHP, two other nuclear receptors previously
implicated in bile acid repression of CYP7A1 expression
(116, 230). Together these studies suggest that SXR and its
rodent counterpart, PXR, may serve as functional bile salt
receptors and facilitate upregulation of CYP3A, thereby
enhancing metabolism of potentially toxic bile salts.
While studies of SXR and PXR expression will be necessary in patients and animal models with cholestatic liver
injury, the antibiotic rifampicin, another potent ligand for
649
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MICHAEL TRAUNER AND JAMES L. BOYER
promoters of CYP2B and CYP3A with DR-3, D-4, or
everted repeats with a 6-bp spacer. Thus these two signaling pathways appear to interact in the regulation of
xenobiotic drug metabolism (346). FXR also binds with
high affinity with RXR-␣ to response elements in the rat
Mrp2 promoter that are shared with CAR and PXR (172).
Thus bile salts as well as CAR and PXR ligands can
regulate Mrp2 expression, since both naturally occurring
(CDCA) as well as synthetic (GW4064) FXR ligands induce the expression of MRP2/Mrp2 mRNA in human and
rat hepatocytes (172). Moreover, CAR activators have
been shown to induce Mrps1–3 in rat liver but not in
extrahepatic tissues such as kidney and intestine (65).
A) AP-1. Two AP-1 consensus sites have been identified in the proximal promoter of the rat ileal apical
sodium-dependent bile salt transporter Isbt. Both c-jun
and c-fos bind to these elements and enhance promoter
activity in transfected cell lines (92).
B) HNF-1␣. HNF-1␣ is a “liver-enriched” transcription factor that regulates transcription of several hepatocyte-specific genes including the bile salt transporter Ntcp
and plasma proteins such as albumin, ␣1-antitrypsin, fibrinogen, etc. (for review, see Ref. 360). Two isoforms
have been identified: HNF-1 (or HNF-1␣) and vHNF (or
HNF-1␤). HNF-1␣ is predominant in hepatocytes but is
also present in epithelial cells of other organs including
renal proximal tubules, stomach, small intestine, colon,
and pancreas, whereas HNF-1␤ has an even wider distribution. Both factors can form homo- and heterodimers
that bind to a 15-nucleotide-long consensus sequence
with a palindrome structure (A/GTTAAT). HNF-1␣ homodimers are the major transcription factors in hepatocytes. HNF-1␣ is also regulated by HNF-4, another liverenriched transcription factor (200). HNF-1␣ negatively
regulates its own expression and that of other HNF-4dependent genes by negative feedback inhibition of the
main activation domain of HNF-4 (200). HNF-1␣ plays an
important role in the constitutive expression of Ntcp (81,
170, 356). Expression of Ntcp, Oatp1, and Oatp2 are significantly reduced in the livers of HNF-1␣ knock-out mice
(318), and Isbt is not expressed in the terminal ileum,
leading to fecal bile salt loss. HNF-1␣ also has a direct
role in activating transcription of the Isbt gene (318).
HNF-1␣ also regulates the expression of the nuclear bile
acid receptor FXR/BAR (318), which in turn plays a critical role in the transcriptional regulation of Bsep, SHP,
and I-Babp.
Human HNF-1␣ is required for the expression of
OATP-C (SLC21A6) as well as OATP8 (SLC21A8) and
mouse Oatp4 (Slc21a6) (161). Thus HNF-1␣ seems to play
Physiol Rev • VOL
B. Posttranscriptional Regulation
of Bile Salt Transporters
There is considerable evidence that several bile salt
transporters in hepatocytes are regulated posttranscriptionally and that their cell surface expression can be
stimulated by recruitment from intracellular pools (183).
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2. Role of other transcription factors in regulation
of bile salt transporters
a major role in bile salt transporter expression and homeostasis.
C) HNF-3␤. Transgenic mice that overexpress
HNF-3␤ have reduced hepatic levels of expression of Ntcp
and Mdr2 (291). This finding could be explained by studies demonstrating that HNF-3␤ transactivates the liverenriched homeobox gene (Hex), which in turn positively
regulates the Ntcp promoter (84).
D) HNF-4. Recent studies in conditional gene knockout mice for HNF-1␣ and HNF-4␣ report reduced levels of
basolateral bile salt and organic anion uptake systems
(Ntcp, Oatps), indicating a central role to these transcription factors in maintaining basolateral transporter expression (99, 133, 175). Part of these effects could indirectly
be explained by HNF-4 effects on HNF1 expression (200).
E) Signal transducers and activators of transcription. Signal transducers and activators of transcription
(STATs) consist of a family of signal transduction proteins activated by binding of extracellular polypeptides
(growth factors and cytokines) to transmembrane receptors (for review, see Ref. 141). STAT5 activates the rat
Ntcp promoter by binding to two closely spaced consensus sites that may explain upregulation of Ntcp expression by growth hormone and prolactin (103).
F) C/EBPs. C/EBPs are a family of leucine zipper
transcription factors involved in the regulation of various
aspects of cellular differentiation and proliferation, metabolic function (e.g., gluconeogenesis, carbohydrate metabolism), and response to inflammatory insults in hepatic, adipose, and hematopoietic tissues (288; for review,
see Ref. 224). The human NTCP and rat Ntcp promoters
contain several putative C/EBP-␣ and -␤ binding sites (81,
170, 317). A C/EBP element is required for maximal retinoid transactivation of the rat Ntcp promoter by RXR-␣:
RAR-␣ (81). Further putative C/EBP binding sites have
also been identified in the rat Mdr1b and human MDR3
and MRP2 promoters (257, 340), suggesting that this transcription factor could play an important role in the regulation of hepatobiliary transporter expression.
G) PPAR-␣. Recent studies have suggested a physiological role for PPAR-␣ in mediating human ISBT gene
regulation (160). This is the first evidence that PPAR-␣ is
involved in bile salt homeostasis by regulating intestinal
bile salt uptake.
BILE SALT TRANSPORTERS
1. Hepatocellular basolateral transporters
2. Hepatocellular canalicular transporters
Current evidence suggests that several members of
the superfamily of ABC transporters that are functionally
expressed at the apical canalicular domain reside within
submembranous endosomal pools that can be mobilized
in response to several stimuli (Fig. 3) (45, 184, 185, 296).
Studies in the isolated perfused rat liver indicate that
infusions of cAMP increase the biliary excretion of bile
salts by stimulating the intracellular vesicle transport system (131) and that simultaneous infusions of taurocholate
further augment secretion of phospholipids and bile salts
(132). Administration of dibutyryl cAMP to rat hepatocyte
Physiol Rev • VOL
couplets results in expansion of the apical domain as well
as increased and enhanced canalicular excretion of fluorescent Mrp2 substrates. These findings were associated
with enhancement of apical membrane expression of
Mrp2 as assessed by immunofluorescence staining (296).
Dibutyryl cAMP treatment also stimulates excretion of
bile salt fluorescent substrates in hepatocyte couplets
(45). The effects of cAMP are acute and thus not dependent on protein synthesis. Inhibitors of microtubule function also block these effects, supporting a role for microtubules in this regulatory process.
Studies in the intact rat, using intravenous administration of dibutyryl cAMP as well as taurocholate, also
demonstrate a rapid and selective increase in Mdr1 and
Mdr2 as well as Mrp2 and Bsep in canalicular membrane
vesicles isolated from these livers. This finding correlates
functionally with increased ATP-dependent transport of
substrates for these apical membrane transporters (109).
This process is inhibited by colchicine as well as wortmannin, an inhibitor of PI 3-kinase, supporting a role for
microtubules and PI 3-kinase in this process. Cycloheximide treatment has no effect on these phenomena, emphasizing that these are postranscriptional events (248,
249). Additional studies in isolated canalicular membrane
vesicles from rat liver suggest that 3⬘-phosphoinositide
products of PI 3-kinase also have direct posttranslational
effects on Mrp2 and Bsep but not Mdr1 transport activity
(184, 248).
Taurocholate and cAMP appear to stimulate recruitment of ABC transporters from separate pools since simultaneous administration of these two agents augments
the canalicular expression levels of these transporters
above that observed with only a single agonist. Furthermore, these studies suggest that the majority of the bile
salt export pump protein resides within an intracellular,
presumably endosomal compartment, from which they
are mobilized to the canalicular domain in response to
metabolic need (185).
The bile salt tauroursodeoxycholic acid (TUDCA),
which is widely used to improve bile secretory function in
patients with intrahepatic cholestasis, also can stimulate
the insertion of transport proteins into the canalicular
apical membrane under certain physiological and cholestatic conditions. Studies in isolated rat hepatocytes and
perfused livers indicate that TUDCA but not UDCA is
capable of stimulating canalicular exocytosis by producing a sustained elevation in intracellular calcium in the
cytosol (30, 31). This process involves depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive microsomal Ca2⫹
stores by an IP3-independent mechanism that leads to
mobilization of extracellular sources of calcium through
Ni2⫹-sensitive Ca2⫹ channels in the plasma membrane
(31). This sustained increase in intracellular calcium is
associated with translocation of the Ca2⫹-sensitive isoform, ␣-PKC, to the plasma membrane, a process that
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Studies in isolated rat hepatocytes indicate that
cAMP stimulates sodium bile salt cotransport via a protein kinase A-mediated event that can be potentiated by
increases in intracellular calcium and downregulated by
activation of protein kinase C (120). This stimulatory
effect of cAMP is associated with increased content of
Ntcp in the plasma membrane and a decrease of Ntcp in
endosomal compartments and does not involve protein
synthesis (254). Subsequent studies showed that Ntcp is a
serine/threonine phosphoprotein and that cAMP results in
Ntcp dephosphorylation (255), possibly leading to retention of Ntcp in the plasma membrane and subsequent
stimulation of Ntcp-mediated bile salt uptake. Further
studies indicate that okadaic acid, an inhibitor of protein
phosphatase 2A, prevents cAMP-stimulated translocation
of Ntcp in part by decreasing cAMP-induced increases in
cytosolic Ca2⫹. However, okadaic acid increased Ntcp
phosphorylation without inhibiting basal taurocholate uptake. Therefore, increased phosphorylation due to inhibition of protein phosphatase 2A does not affect Ntcp activity. These studies suggest that cAMP-mediated signal
transduction is maintained by protein phosphatases, but it
remains unclear whether cAMP-mediated dephosphorylation is due to inhibition of a kinase or activation of other
protein phosphatases (255). Inhibitors of phosphatidylinositol (PI) 3-kinase and actin filament formation also
block the ability of cAMP to translocate Ntcp to the
plasma membrane and increase taurocholate uptake in
rat hepatocytes (90, 378, 379). Oatp1 activity also can be
mediated by alterations of its phosphorylation (114).
Phosphorylation of specific tyrosine residues on the
cytoplasmic tail of rat Ntcp appears to be involved in
targeting of this basolateral transporter to this specific
plasma domain (347). Truncation of the 56-amino acid
cytoplasmic tail inhibits delivery of a green fluorescent
protein (GFP)-Ntcp construct to the plasma membrane in
C0S-7 and MDCK cells. Two tyrosine residues, Tyr-321
and Tyr-307, have been identified in site-directed mutagenesis experiments, with Tyr-321 representing the major basolateral sorting determinant (347).
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MICHAEL TRAUNER AND JAMES L. BOYER
facilitates exocytosis (33). Studies in isolated perfused rat
livers demonstrate that TUDCA can also reverse the cholestatic effects of taurolithocholate (31). TLCA translocates the calcium-insensitive isoform protein kinase C-⑀
to the canalicular membrane in isolated rat hepatocytes, a
process that is also reversed by TUDCA (32). Simultaneous infusions of TUDCA with taurolithocholate (TLCA)
can restore the density of Mrp2 transporters on the canalicular membrane and the functional capacity to excrete
2,4-dintrophenyl-S-gluthatione, an Mrp2 substrate, into
bile (29). Treatment of HepG2 cells with phorbol esters,
agonists of protein kinase C, results in loss of Mrp2 from
the apical domain of hepatocyte and relocation to the
basolateral domain, presumably by lateral diffusion via
disrupted tight junctions. The protein kinase C inhibitor
G0 6850 blocked this process (201).
Other studies in rats indicate that TUDCA also stimulates the bile excretory mechanism by enhancing the
canalicular excretion of bile salts (99, 130). This process
is thought to be mediated by an increase in bile salt
excretory capacity, which is regulated by microtubuledependent apical targeting of vesicles that contain the bile
Physiol Rev • VOL
salt transporters and is dependent on signaling pathways
that involve mitogen-activated protein (MAP) kinases
(211, 309). In perfused rat liver, TUDCA activates signaling pathways that involve PI 3-kinase, Ras, a small monomeric GTP binding protein (211, 248), and extracellular
signal-regulated kinase (Erk)-type MAP kinases. These
stimulatory effects of TUDCA on taurocholate excretion
can be blocked by inhibitors of PI 3-kinase. Recent studies suggest that TUDCA-induced stimulation of bile salt
excretion is accompanied by p38 MAP kinase-dependent
insertion of the bile salt export pump, Bsep, from subcanalicular regions into the apical canalicular domain of
hepatocytes (212).
In mice, UDCA feeding results in upregulation of
overall expression of canalicular Bsep and Mrp2 without
any effect on Mdr2 or basolateral Ntcp (99). Both bile salt
and glutathione excretion were also enhanced, indicating
that these findings are functionally relevant. Changes in
overall protein expression may go hand in hand with
stimulation of their insertion into the canalicular membrane. Thus a complex signaling pathway, which is not yet
fully elucidated, appears to be involved in the regulation
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FIG. 3. Posttranscriptional regulation of canalicular transport systems. Canalicular ABC transporters reside within
submembranous endosomal pools that can be mobilized in response to several stimuli such as hyposomolarity, cAMP,
activation of protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K), and mitogen-activated protein kinases
(MAPK). Hyperosmolarity and pathological stimuli such as endotoxin (lipopolysaccharide, LPS)/cytokines, biliary
obstruction (CBDL), phalloidin, and oxidative stress result in retrieval of membrane transporters with a reduction of the
number of functional transporters in the canalicular membrane. In addition to retrieval, impaired targeting with reduced
insertion of transport proteins into the canalicular membrane may also contribute to cholestasis. The retrieved
membrane transporters may initially undergo reinsertion from a subapical vesicular compartment, but later are degraded
by the lysosomal or ubiquitin-proteasome pathway. Posttranslational modifications of transport proteins [e.g., phosphorylation (P), phosphoinositide (PI)-3-kinase products (PI-P1–3)] may rapidly modify transport activity. Cis-inhibition
[from cytoplasmic side, e.g., cylosporin A (CSA) and other drugs] and trans-inhibition [from canalicular side, e.g.,
estradiol 17␤-glucuronide (E217G)] of efflux pumps may be particulary relevant for drug-induced cholestasis.
BILE SALT TRANSPORTERS
III. BILE SALT TRANSPORT DEFECTS
IN CHOLESTATIC LIVER INJURY
As the molecular mechanisms of bile formation and
bile salt transport have become clearer, they have provided additional insight into the mechanisms of cholestasis. However, this pathophysiological process is complex
and may result not only from defects in the regulation and
expression of the membrane transporters that determine
the bile secretory process but also numerous intracellular
events that impair signal transduction pathways, alter the
function of cytoskeletal proteins, affect the expression/
localization of tight and gap junctional proteins, and modify the targeting of intracellular vesicles that maintain cell
polarity. The focus of this section is on the effects of
hereditary and acquired forms of cholestatic liver injury
on the expression of membrane transporters that transport bile salts (see Refs. 41, 154, 220, 258, 358 for additional information on the molecular pathophysiology of
cholestasis).
A. Genetic Defects in Bile Salt Transport Proteins
(FIC1, BSEP, MRP2) and MDR3
Mutations in genes that code for several canalicular
membrane transporters result in inherited progressive
cholestatic liver disorders in infancy known as progressive familial intrahepatic cholestasis (PFIC) (153, 154)
(Fig. 4). Although rare homozygous recessive disorders,
these diseases provide critical information concerning the
important role that these transporters play in the normal
formation of bile and the pathogenesis of cholestasis. One
of these transporters is the canalicular bile salt export
pump (BSEP), another is a phospholipid export pump
(MDR3), whose function is critical to the formation of bile
salt mixed micelles in bile, and the third (FIC1) is without
known function. Mutations in these transporters (FIC1,
BSEP, and MDR3) account for the phenotypic expression
of PFIC-1, -2 and -3.
FIG. 4. Hereditary transport defects
in hepatocytes. Mutations of hepatocellular transport systems can cause congenital cholestasis. Examples include
different forms of progressive familial intrahepatic cholestasis (PFIC1–3,) benign
recurrent intrahepatic cholestasis (BRIC),
and subtypes of intrahepatic cholestasis
of pregnancy. Moreover, mutations of
the phospholipid export pump (MDR3)
may also manifest as cholesterol gallstone disease. Hepatocellular transport
defects can also cause noncholestatic
disorders such as Wilson’s disease (mutations of the canalicular copper export
pump, ATP7B), Dubin-Johnson syndrome (mutations of the canalicular bilirubin conjugate export pump, MRP2),
and sisterolemia (mutations of the sitosterol halftransporters ABCG5/G8). Mutations of cholangiocellular cystic fibrosis
transmembrane conductance regulator
(CFTR) may result in cholestasis frequently associated with cystic fibrosis.
Physiol Rev • VOL
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of intracellular trafficking of the bile salt transporter proteins.
The signals for retrieval of canalicular transport systems have been less well studied. Hyperosmolarity and
pathological stimuli such as endotoxin (lipopolysaccharide, LPS)/cytokines, biliary obstruction (CBDL), phalloidin, and oxidative stress result in retrieval of membrane
transporters with a reduction of the number of functional
transporters in the canalicular membrane (202, 278, 298,
310). In addition to retrieval, impaired targeting with reduced insertion of transport proteins into the canalicular
membrane may also contribute to cholestasis. The retrieved membrane transporters may initially undergo reinsertion from a subapical vesicular compartment, but the
latter are degraded by the lysosomal or ubiquitin-proteasome pathway (for review, see Refs. 184, 185) (Fig. 3).
Anchoring proteins (e.g., radixin), which cross-link transporters with actin filaments, are essential for proper localization of transporters as suggested by defective bilirubin excretion and loss of Mrp2 from the canalicular
membrane in radixin-deficient mice (182).
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MICHAEL TRAUNER AND JAMES L. BOYER
1. Fic1/FIC1
2. Bsep/BSEP
Mutations in the canalicular membrane bile salt export pump (BSEP), formerly known as the sister of Pglycoprotein, account for the syndrome of PFIC-2 (344).
This important discovery provides compelling evidence
for the role of this member of the ABC gene family in
determining bile salt-dependent bile flow. More than 30
mutations have now been described in families with this
familial disorder (52). Patients with PFIC-2 resemble
Byler’s disease phenotypically, but these families are unrelated to the Amish Byler family. PFIC-2 has been described in families from Saudi Arabia and Europe. A
recent report indicates that BSEP mutations may also
present as a compound heterozygote in an adolescent
Physiol Rev • VOL
3. Mdr2/MDR3
Patients with PFIC-3 have mutations in MDR3 that
encode for the phospholipid export pump at the canalicular membrane. This protein functions as a phospholipid
flippase and translocates phosphatidylcholine from the
inner to the outer surface of the canalicular membrane
bilayer. Bile salts are also not substrates for Mdr2/MDR3,
yet when this phospholipid export pump is deficient, bile
salts do not form micelles and can result in damage of
cholangiocytes and progressive cholestatic liver injury
(PFIC-3) (80, 87).
The pathophysiological consequences of this genetic
defect were first established in the Mdr2 knock-out mouse
(327). These animals have a complete deficiency of phospholipid in bile and eventually develop a portal inflammatory response and bile ductular proliferation. Fibrosis
ensues, and with time these animals develop a sclerosing
cholangitis and biliary-type cirrhosis and hepatocellular
carcinomas (239, 327). Expression of the human MDR3 in
Mdr2 (⫺/⫺) animals restores the normal phenotype and
the ability of the mice to excrete phospholipid (329).
Transplantation of Mdr3 transgenic hepatocytes into
Mdr2 (⫺/⫺) mice also corrects this abnormality (86).
Lipoprotein-X is an abnormal lipoprotein that accumulates in the circulation in cholestatic liver injury, probably
as a result of the regurgitation of bile into plasma. However, this lipoprotein is absent in the bile duct-obstructed
Mdr2 (⫺/⫺) mouse. These studies indicate that phospholipids must first be excreted into bile for this abnormal
serum lipoprotein to be formed (275).
Several recent studies have reported MDR3 mutations in children and teenagers with progressive cholestasis and intrahepatic gallstones (151). Recent studies have
emphasized the wide phenotypic spectrum of MDR3 deficiency ranging from neonatal cholestasis and cholestasis
of pregnancy to cirrhosis in adults (151). Children with
missense mutations have a less severe disease and more
often benefit from UDCA (151). These mutations led to
truncations and absence of the MDR3 protein at the canalicular membrane in some and to single-base pair
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Homozygous mutations in FIC1 result in PFIC-1 described in an Amish kindred and known as Byler’s disease
(72, 96, 335, 361) and in Inuit populations in Greenland
(187). Mutations in the same genomic regions are also
associated with a rare benign form of intrahepatic cholestasis in adults known as benign recurrent intrahepatic
cholestasis, or BRIC, and found primarily in Scandinavian
families (50, 364). Mutations in this gene result in a profound reduction of bile salts in bile, which is most pronounced for hydrophobic toxic bile salts such as lithocholate and chenodeoxycholate. PFIC-1 is a progressive
ultimately fatal pediatric cholestatic disorder characterized by normal ␥-glutamyltransferase levels in the serum,
the absence of bile duct proliferation on liver histology, as
well as frequent extrahepatic manifestations such as diarrhea, malabsorption, and pancreatitis. Electron microscopic studies reveal highly characteristic lesions of the
canalicular membrane as well as accumulation of intracellular membrane components within the canalicular lumen known as “Byler’s bile.” Symptoms of diarrhea persist after liver transplantation, indicating a more general
role of FIC1 in the (intestinal) defense against toxic bile
salts. In line with such a hypothesis, preliminary/recent
findings indicate that the FIC1 knock-out mouse accumulates bile salts in serum while maintaining normal bile salt
excretion, suggesting that the primary defect is in regulating intestinal bile salt absorption (283).
It remains to be determined how mutations in the
same regions of the gene can result on the one hand in a
progressive fatal cholestatic disorder of infancy while on
the other account for a benign recurrent cholestatic process in adults. Equally unclear is the finding that some
members of BRIC families have mutations that result in
cholestatic disease while others with similar mutations do
not express the cholestatic phenotype. These apparent
paradoxes suggest that other genetic and/or environmental factors may be required as a “second hit” for the
cholestatic phenotype to be expressed.
with BRIC (209), suggesting that different mutations may
account for differences in severity of the disease. Again, it
is likely that environmental factors also influence the
phenotypic expression of these mutants.
A recent study has examined the effect of seven of
the PFIC-2 associated missense mutations when introduced into rat Bsep and expressed in MDCK and Sf9 cells
(375). G238V, E297G, G982R, R1153C, and R1268Q mutations prevent the protein from trafficking to the apical
membrane, whereas the G238V mutant seems to be rapidly degraded by proteasomes. Most but not all of these
mutations (C336S and D482G) also abolish bile salt transport activity when assessed in Sf9 cells (54, 314, 375, 376).
BILE SALT TRANSPORTERS
4. Mrp2/MRP2
The mutant TR⫺/GY/EHBR rats have mutations in
the Mrp2 gene that result in a stop codon and premature
termination of protein translation (171, 282, 369). In humans, mutations in MRP2 produce the Dubin-Johnson
syndrome, a cause of hereditary conjugated hyperbilirubinemia. Mutations in this gene in the rat model and
humans result in defects in the excretion of a variety of
amphipathic organic anions, including divalent bile acids,
conjugated bilirubin, coproporphyrin isomer series 1, leuPhysiol Rev • VOL
kotrienes, as well as many other compounds such as BSP,
indocyanin green, oral cholecystographic agents, antibiotics such as ampicillin and ceftriaxone, and heavy metals. When several of the MRP2 mutant genes described in
Dubin-Johnson syndrome are expressed in HepG2 cells,
the mutated protein is unable to be target to the apical
membrane but is retained within the endoplasmic reticulum and subsequently degraded in proteosomes (177).
GSH, oxidized GSSG, and GSH conjugates are endogenous substrates for Mrp2. GSH is a low-affinity substrate
for Mrp2 and a major determinant of bile salt-independent
bile flow that is significantly reduced in the TR⫺/GY/
EHBR rat model (157). It is likely that polymorphisms in
Mrp2/ MRP2 and agents that impair the Mrp2/MRP2 transporter expression and function will diminish GSH excretion and lead to cholestatic injury. In addition, such mutations could also indirectly result in cholestasis through
impaired excretion of potentially cholestatic metabolites
of drugs and hormones. Mrp3/MRP3 expression is also
increased after common bile duct ligation in the rat and in
hepatocytes in patients with the Dubin-Johnson syndrome and the TR⫺/GY/EHBR rat where canalicular expression of Mrp2 is genetically absent (136, 192). These
responses may be viewed as a protective adaptation in
situations where MRP2/Mrp2 function is impaired (89).
B. Acquired Defects in Bile Salt Transport
Proteins in Cholestasis
Studies in several experimental models of cholestasis
including common bile duct ligation (CBDL), estrogen/
ethinyl estradiol (EE), and endotoxin (LPS) administration as well as more limited studies in acquired cholestatic diseases in humans provide information on the effects of this form of liver injury on the expression of bile
salt transport proteins in the liver and other tissues (for
review, see Ref. 220). In general, there is a common
pattern of responses that serves to partially protect the
hepatocyte from the retention of toxic bile salts, but also
contributes to/aggravates systemic bile salt retention (Table 3, Fig. 5). The transport of bile salts from portal blood
into hepatocytes is diminished by downregulation of several bile salt transporters on the basolateral membrane
including Ntcp, Oatp1, and r-Lst1 (incomplete splice isoform of Oatp4) but not Oatp2 and Oatp4. At the same
time, bile salt export mechanisms in hepatocytes continue
to be expressed (Bsep) and in some instances may be
upregulated (Mrp3) (89, 331). Mrp3 should be able to
transport bile salts back into the systemic circulation,
since Mrp3 has a high affinity for lithocholate sulfate and
other bile salt conjugates that accumulate in the cholestatic liver (138).
Cholangiocyte bile salt transporters are also affected
during cholestasis (Table 3, Fig. 5). Bile ducts proliferate
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changes in others. Only individuals with the latter defect
(missense mutations) responded clinically to treatment
with the bile salt UDCA, probably because the protein
could still be expressed. Mdr2 heterozygote (⫹/⫺) mice
excrete half the normal level of phospholipids into bile
but are phenotypically normal. Mothers of homozygous
MDR3-deficient children with PFIC-3 are also obligate
heterozygotes. This partial defect in phospholipid excretion predisposes them to the development of cholestasis
during the third trimester of pregnancy when estrogen
levels rise and further impair bile secretion (150). MDR3
deficiency may account for one-third of patients with high
GGT-PFIC (PFIC-3) in European patients (151) but only
2% of Taiwanese infants with this syndrome (64), suggesting a role for additional gene defects. Hereditary MDR3
defects are also involved in intrahepatic and gallbladder
cholesterol gallstone formation and the pathogenesis of
cholestasis of pregnancy (67, 151). MDR3 deficiency may
be linked to cholesterol gallstone formation through defective biliary excretion of phospholipids resulting in cholesterol supersaturation of bile. Reduced MDR3 levels
have also been found in oriental patients with intrahepatic
bile duct stones, suggesting a role for heterozygotes with
MDR3 defects in the pathogenesis of this syndrome (324).
These uncommon disorders emphasize the importance of the normal function of the phospholipid export
pump and the relevance of mutations in this transporter
to the pathogenesis of certain cholestatic diseases. Polymorphisms in the expression of MDR3 might predispose
individuals to cholestatic liver injury, particularly if they
become exposed to drugs or environmental toxins that
are potential cholestatic agents. A few studies in patients
with primary biliary cirrhosis and alcoholic cholestatic
liver disease report normal MDR3 mRNA levels (93, 401,
403). The only acquired disease with abnormal (reduced)
biliary phospholipid excretion identified in adults so far is
TPN-induced cholestasis, although no information on
MDR3 expression data is available so far (88). Sclerosing
cholangitis-like changes in Mdr2 (⫺/⫺) mice (99a, 239)
suggest that MDR3 defects could be involved in adults
with PSC. Further studies are needed to determine the
role that MDR3 mutations and polymorphisms play in
idiosyncratic cholestatic disorders in the adult.
655
656
TABLE
MICHAEL TRAUNER AND JAMES L. BOYER
3. Changes in transporter expression during acquired cholestasis and proposed mechanism of regulation
Transporter
Change in Expression During Cholestasis
Proposed Mechanisms*
Basolateral membrane of hepatocyte
Ntcp/NTCP
Downregulation
Oatps/OATPs
Mrp3/MRP3
Downregulation of rodent Oatp1 and human OATP2
Highly upregulated on basolateral membrane of hepatocytes
in cholestasis
1) Bile salt binding to FXR stimulates expression of SHP, 2)
loss of vitamin A (RAR ligand), and 3) IL-1␤-mediated
downregulation of RXR/RAR NHR; all 3 effects may
diminish Ntcp gene expression 4) loss of HNF-1␣ activity
Loss of HNF-1␣
Transcriptional regulation in hepatocytes not yet determined
Canalicular membrane of hepatocyte
Upregulated in most forms of cholestasis
Mdr2/MDR3
Mrp2
Bsep/BSEP
No change in expression
Downregulation in most forms of cholestasis (human MRP2
well maintained)
Partially downregulated
FIC1
Unchanged
IL-1␤-mediated downregulation of RXR/RAR NHR that
regulate Mrp2 transcription
Hydrophobic bile salts bind to FXR, resulting in continued
transcription with partial recovery of Bsep/BSEP
Cholangiocytes
Isbt
Expression maintained after common bile duct obstruction
CFTR
AE2
Not known
Continues to be expressed in bile duct proliferation after bile
duct obstruction, reduced in PBC
Continues to be expressed after common bile duct ligation in
rodents and PBC in humans
Mrp3/MRP3
Not known but may be related to bile salt binding to FXR
resulting in upregulation of the ileal-bile acid binding
protein that associates with Isbt
Not known
Not known
Intestine
Oatp3
Isbt
Mrp3/MRP3
Not known
Downregulated after common bile duct obstruction
Continues to be expressed after common bile duct
obstruction
Not known
Not known
Kidney
Isbt
Mrp2
Downregulated after common bile duct ligation
Upregulated by posttranscriptional mechanisms after
common bile duct ligation
Not known
Not known
Data are from animal models and cholestasis in patients. PBC, primary biliary cirrhosis; RAR, retinoic acid receptor; FXR, farnesoid X receptor;
SHP, short heterodimeric protein; IL, interleukin; RXR, retinoid X receptor; NHR, nuclear hormone receptor; HNF, hepatocyte nuclear factor.
* Most of these proposed mechanisms of regulation in cholestasis remain to be confirmed by in vivo studies in cholestatic animal models and
in humans and are inferences based on in vitro studies and knowledge of their physiological regulators.
in most cholestatic conditions, resulting in a marked increase in the number of cholangiocytes. The ileal sodiumdependent bile salt transporter (Isbt), located on the luminal membrane of cholangiocytes, may function to remove bile salts from bile if the cholestatic hepatocyte
continues to excrete bile salts. Mrp3 is located on the
basolateral membrane of the cholangiocyte and continues
to be expressed on proliferating cholangiocytes (331). As
a result of this proliferative activity, the total amount of
this transporter is upregulated in the cholestatic liver.
Changes in the expression of several renal and intestinal bile salt transporters (Ibst and Mrp2) also take place
during cholestasis, thereby facilitating the urinary and
Physiol Rev • VOL
intestinal excretion of bile salts and bilirubin (219, 352).
Altogether, these changes in expression of liver, kidney,
and intestinal bile salt transporters can be interpreted as
an attempt to mitigate tissue damage from hepatic and
systemic retention of bile salts (Table 3, Fig. 5).
Adaptive regulation of the expression and function of
these membrane transporters occurs by several different
mechanisms, including 1) alterations in gene transcription mediated by elements that interact with regulatory
elements in gene promoters as described in more detail in
section II (Table 3); 2) posttranscriptional changes in
mRNA processing or message stability; or 3) posttranscriptional changes that result either in defects in protein
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Cytokine-mediated nuclear translocation of NF␬B
upregulates Mdr1b expression
Mdr1b/MDR1
BILE SALT TRANSPORTERS
657
trafficking or the regulation of the proteins activity though
phosphorylation/dephosphorylation reactions or direct
inhibition by substrates/compounds (Fig. 3). Because the
half-life of these membrane transporters is several days,
acute changes in the activity of transporter function are
likely to occur by a posttranscriptional mechanism,
whereas transcriptional regulatory responses would be
expected in chronic adaptive responses.
1. Effects of cholestasis on basolateral membrane bile
salt transporters in hepatocytes (Ntcp /NTCP,
Oatps/OATPs, Mrp1/MRP1, Mrp3/MRP3)
A) Ntcp/NTCP. This transporter is uniformly downregulated in all experimental models of cholestasis and in
cholestatic liver disease in humans (220). Nuclear run-on
studies in the CBDL and LPS-treated rat indicate that this
downregulation occurs at least in part at the level of gene
transcription (106, 356). After inhibition of gene transcription, Ntcp protein disappears rapidly from the sinusoidal
membrane throughout the hepatic lobule. In children with
biliary atresia, Ntcp mRNA is diminished but increases if
biliary drainage is restored by a successful portoenterosPhysiol Rev • VOL
tomy (Kasai procedure) (321). Levels of hepatic Ntcp
mRNA correlate inversely with bile salt levels in bile
duct-obstructed rats and humans, respectively (108), and
with levels of total serum bilirubin in patients with biliary
atresia (321). These changes in Ntcp expression are not
reproduced by choledochocaval fistulas or by partial bile
duct ligation, suggesting that bile retention rather than the
magnitude of bile salt flux is important in regulation of
Ntcp gene transcription (108). However, cholate feeding
to mice reduces expression of Ntcp, suggesting that endogenous bile salts retained during cholestasis could also
be involved in the regulation of Ntcp gene expression (99,
326). The promoter of Ntcp in the rat contains several
regulatory elements including HNF-1, as well as an RXR/
RAR retinoic acid element. Bile salt-induced SHP-1 inhibits retinoid transactivation (via RXR:RAR) of the rat Ntcp
promoter in vitro (85) (Table 3). Along these lines, bile
duct ligation in mice induces SHP-1 expression in parallel
with accumulation of serum bile salt levels and precedes
downregulation of Ntcp by several hours (402). Cholestatic doses of LPS result in loss of HNF-1 and RXR:RAR
activities in rat liver and decrease Ntcp expression in
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FIG. 5. Hepatic and extrahepatic adaptive changes in hepatobiliary transporter expression in animal models of
cholestasis. Downregulation of the basolateral uptake systems such as the Na⫹-taurocholate cotransporter (Ntcp) and
organic anion transporting protein 1 (Oatp1), together with partial recovery of the canalicular bile salt export pump
(Bsep) with prolonged duration of cholestasis reduces the hepatocellular retention of bile salts (BS⫺). In contrast to
downregulation of the canalicular conjugate export pump (Mrp2), expression of another isoforms of the multidrug
resistance-associated protein (Mrp1 and Mrp3) increases at the (baso)lateral membrane of hepatocytes, providing an
alternative route for the elimination of BS⫺ and nonbile salt organic anions (OA⫺) from cholestatic hepatocytes into the
systemic circulation. In contrast to hepatic downregulation of Mrp2 and upregulation of the cholangiocellular apical/ileal
Na⫹-dependent bile salt transporter (Isbt), renal Mrp2 is upregulated while Isbt is downregulated in the kidney. These
reciprocal changes may help the renal elimination of BS⫺ and conjugated bilirubin in cholestasis. Increased expression
of the cholangiocellular and Mrp3 (and possibly a truncated basolateral Isbt isoform; t-Isbt) facilitates the return of BS⫺
from obstructed bile ducts to the systemic circulation followed by their renal excretion. Enterohepatic recirculation of
BS⫺ is impaired as a result of reduced hepatic excretion and decreased expression and function of ileal Isbt.
658
MICHAEL TRAUNER AND JAMES L. BOYER
Physiol Rev • VOL
under cholestatic conditions remain to be determined
(210).
C) Mrp1/MRP1, Mrp3/MRP3. Mrp1 is substantially
upregulated during cholestasis produced by LPS (367),
while Mrp3 is induced in common bile duct obstruction
but not in EE-induced cholestasis. Upregulation of MRP3
is also seen in patients with obstructive cholestasis (323)
and PBC (401). Studies in the rat indicate that Mrp3
expression is normally expressed only on pericentral venous hepatocytes but is progressively upregulated after
CBDL so that by 14 days, Mrp3 is expressed throughout
the hepatic lobule (331). This increase in Mrp3 parallels
the progressive downregulation of the canalicular homolog Mrp2. Mrp3/MRP3 expression is also increased in
hepatocytes in patients with the Dubin-Johnson syndrome and the TR⫺/GY/EHBR rat where canalicular expression of Mrp2/MRP2 is genetically absent (136, 192,
282, 369). In addition, Mrp3 is also upregulated in FXR
(⫺/⫺) mice with reduced canalicular Bsep levels (314).
These observations suggest that Mrp3 may facilitate efflux
of substrates into sinusoidal blood that normally would be
extruded into bile by Mrp2/MRP2 (314). Because sulfated
bile salt conjugates are synthesized in the cholestatic liver
and are high-affinity substrates for Mrp3 as demonstrated
in vitro in cellular expression systems (138), we speculate
that Mrp3/MRP3 may function as an important efflux
pump in cholestasis, thereby protecting hepatocytes from
the accumulations of toxic levels of bile salts (138). In
summary, upregulation of basolateral MRP3 during cholestasis may not only explain the appearance of conjugated bilirubin in plasma, but may also provide an alternative efflux route for bile salts from hepatocytes into the
sinusoidal blood which together with downregulation of
basolateral uptake systems (NTCP and OATP2) prevents
further accumulation of potentially toxic biliary constituents, particularly bile salts within cholestatic hepatocytes
(219). Recent data suggest that the inverse changes of
reduced NTCP and induced MRP3 expression spatially
and temporally coincide and are most pronounced in the
periphery of liver lobules and cirrhotic nodules in PBC
livers (401).
2. Effects of cholestasis on bile salt transporters
on the canalicular membrane of the hepatocyte
(Bsep/BSEP, Mrp2/MRP2, Mdr2/MDR3, Fic1/FIC1)
A) Bsep/BSEP. In contrast to downregulation of Ntcp
and Mrp2 in animal models of cholestasis (LPS, EE,
CBDL), Bsep expression is only modestly impaired and
bile salt excretion continues, albeit at reduced levels,
even in the face of complete bile duct obstruction (221).
In contrast, many cholestatic agents, including cyclosporin A, glibenclamide, rifamycin, rifamapin (336),
triglitzone (102), and bosentan (97) cis-inhibit ATP-dependent taurocholate transport in vitro in isolated rat liver
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parallel with reductions in bile flow (356) (Table 3). Reductions in RXR mRNA levels are also part of the acute
phase response. In hamster liver, LPS and proinflammatory cytokines, including interleukin-1␤ and TNF-␣, induces a rapid dose-dependent decline in three distinct
RXR mRNAs and proteins RXR-␣, -␤, and -␥ within several
hours (24). Stress pathway activation induces phosphorylation of RXR via both mitogen-activated protein kinase
kinase-4 and its downstream mediator JNK, resulting in
reduced RXR activity (223, 226). Finally, LPS also reduced
HNF-1 activity (356), which in turn may be secondary to
reduced HNF-4 activity (372) that controls HNF1 gene
transcription. Furthermore, it is speculated that depletion
of vitamin A stores in the liver may contribute to reduced
expression of genes containing RXR/RAR response elements. Vitamin A stores are diminished in cholestatic liver
diseases (269) and could influence the reduced expression of Ntcp and Mrp2 observed in models of cholestatic
liver injury. Finally, preliminary studies indicate that
CBDL in the rat results in rapid loss of RXR, RAR-␣, and
SHP nuclear protein and that these acute effects may be
mediated in part by cytokines, particularly interleukin-1␤
(36, 82).
B) Oatp1, -2, -4/OATP-A and -C. Studies in rat models of CBDL, EE, and LPS administration indicate that all
of these cholestatic models result in downregulation of
Oatp1 (220). mRNA levels remain unchanged after EE
treatment, suggesting that EE administration downregulates Oatp1 mainly by posttranscriptional mechanisms
(325). The expression of Oatp2, which is higher in the
brain than the liver, and Oatp4 is not reduced after CBDL
in the rat (107). This finding is in contrast to the expression of the liver-specific transporter (rLst1), the incomplete splicing isoform of Oatp4, which is markedly diminished after CBDL in the rat (164). In contrast to these
findings, clinical studies in patients with primary sclerosing cholangitis indicate that OATP-A mRNA expression is
increased, suggesting that OATP-A may be able to function in reverse in cholestasis and help to extrude organic
anions from the cholestatic hepatocyte (204). However,
OATP-C is reduced in patients with alcohol and inflammation-induced cholestatic liver injury (40, 403) and primary sclerosing cholangitis (PSC) (270) and primary biliary cirrhosis (PBC) (401). Further studies will be necessary to clarify the functional implications of these
changes in expression of Oatp/OATPs in cholestasis considering the considerable overlap in substrates. Preservation of some Oatps/OATPs under cholestatic conditions
(e.g., rat Oatp2 and -4 in CBDL rats; human OATP-A in
PSC) could mediate monovalent bile salt efflux by reversal of the transport direction, although this remains highly
speculative. The relative contributions of maintained
Oatps/OATPs and upregulated Mrps/MRPs to basolateral
efflux of monovalent and divalent bile salt conjugates
BILE SALT TRANSPORTERS
Physiol Rev • VOL
also inhibit the bile salt export pump if they accumulate in
the liver. Interestingly, unlike most basolateral membrane
transporters that are downregulated in response to cholestatic liver injury, the molecular expression of P-glycoprotein-170 (Mdr1/MDR1) is upregulated in both animal
models of cholestasis and in cholestatic disorders in humans (220). Cholestasis induced by CBDL, LPS, or
␣-napthylisothianate (ANIT) all increase the expression
of Mdr1 mRNA (313, 357, 367). The level of expression
correlates with the severity of the cholestasis, as manifested by the height of serum levels of plasma bilirubin
and alkaline phosphatase (313). Levels of MDR1 in biopsies from patients with bile duct obstruction, inflammatory cholestasis (403), and PBC reveal similar findings
(266, 401). Exogenous stimuli including heat shock, ultraviolet light, chemotherapeutic agents, carbon tetrachloride, and carcinogens also increase the transcription of
the Mdr1 gene promoter (261). Recent studies suggest
that Mdr1 can be upregulated through the action of cytokines such as TNF-␣, which initiates a signal transduction
cascade that results in translocation of NF␬B to the nucleus and stimulation of Mdr1 gene transcription (297).
Unlike other canalicular export pumps, genetic defects in
hepatic MDR-1 P-glycoprotein (170) have not been identified in humans. Bile flow also remains normal in knockout mouse models of Mdr1a and Mdr1b, and organic
cation excretion is only modestly impaired. However,
organic cation transport is significantly impaired in double knock-out [Mdr1a,b (⫺/⫺)] mice despite normal bile
production (328).
Many drugs (nearly 80% by some estimates) are organic cations and are substrates for Mdr1/MDR1. Thus it
is possible that polymporphisms in the expression of
Mdr1/MDR1 might diminish the capacity of the liver to
excrete these substrates and lead to increased retention
of these compounds in the hepatocyte (35). Cationic
drugs normally excreted via Mdr1/MDR1 such as cyclosporin A, rifampin, and rifamycin competitively inhibit the
activity of the bile salt export pump both in isolated rat
canalicular membrane vesicles (336) and when expressed
in Sf9 insect cells (336). Whether polymorphisms in MDR1
lead to hepatic retention of these cationic drugs and
inhibition of Bsep in human liver remains to be established.
D) Mdr2/MDR3. Most studies indicate that the expression of Mdr2/MDR3 is not altered in acquired forms of
cholestasis in humans and rats (41, 220). However, polymorphisms in the expression of MDR3 may predispose to
acquired cholestatic disorders as discussed in the genetics section.
E) Fic1/FIC1. This transporter is unchanged in experimental (CBDL) and acquired human cholestasis (96,
335, 403).
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canalicular membrane vesicles. Some compounds (estradiol 17␤-glucuronide) trans-inhibit Bsep in rat liver only
after excretion into bile as evidenced by the absence of
cholestatic effect in TR⫺/GY/EHBR mutants which lack
Mrp2 (143). This estrogen metabolite also fails to inhibit
Bsep when Bsep is expressed in an Sf9 insect cell line,
unless Mrp2 is also coexpressed (336). Both murine and
human Bsep/BSEP contain FXR response elements in
their promoter, suggesting that increases in liver levels of
bile salts that are specific ligands for FXR may help to
maintain Bsep expression during cholestatic liver injury.
Bsep protein expression is also maintained in normal
amounts and location in clinical studies in patients with
biliary atresia (188) and PBC (401). Together these observations suggest that expression levels of members of the
Mdr/MDR P-glycoprotein family (Bsep, Mdr1, and Mdr3;
see below) are relatively well preserved during cholestatic liver injury, compared with other canalicular proteins such as Mrp2 or the basolateral bile salt transporters
Ntcp and Oatp1.
B) Mrp2/MRP2. Expression of Mrp2 at the mRNA
and protein level is markedly downregulated in all experimental animal models of cholestasis including CBDL, EE,
and LPS (220). Impairment of Mrp2 expression and transport function in endotoxin models of cholestasis provides
an explanation for why sepsis commonly results in elevation of serum levels of conjugated bilirubin (357). Reduced Mrp2 mRNA expression may be explained through
impaired retinoid transactivation via RXR:RAR, which
may be inhibited via bile acid-induced SHP-1 or cytokinemediated downregulation of RXR transcription and RXR
phosphorylation. Reductions in both liver RXR-␣ and RAR
RNA, nuclear protein levels, and DNA binding to the Mrp2
promoter cis-levels in liver contrast with their maintenance of expression in the kidney and may account for
the organ-specific adaptive regulation seen following
CBDL in the rat (36, 82, 83). Nevertheless, studies of
MRP2 expression in patients with cholestatic alcoholic
hepatitis suggest that MRP2 mRNA is not significantly
reduced in this inflammatory disorder (403). In line with
these observations in vivo, LPS also fails to downregulate
MRP2 expression in human liver slices despite being biologically active as indicated by induction of nitric oxide
and proinflammatory cytokines (95). Similar observations
have been made in late-stage PBC (401). These findings
emphasize that observations made in cholestatic animal
models may not necessarily always apply to cholestatic
disorders in humans. However, recent findings that FXR
response elements are contained within the MRP2 promoter suggest that bile salts should be capable of upregulating this transporter in humans (172).
C) Mdr1a,b/MDR1. While bile salts are not nominal
substrates for this P-glycoprotein, and no mutations have
been described to date that result in cholestatic liver
disease, certain drugs and substrates for Mdr1/MDR1 may
659
660
MICHAEL TRAUNER AND JAMES L. BOYER
3. Adaptive responses of bile salt transporters
in cholangiocytes
4. Adaptive responses of bile salt transporters
in kidney
Bile salt secretion in urine is markedly increased in
both cholestatic animal models and in clinical cholestatic
disorders (71, 219, 338, 388). Recent studies in bile ductligated rats indicate that Isbt mRNA and protein are
downregulated, resulting in reduced levels of expression
on the luminal membrane of the proximal tubule of the rat
kidney, a change that is associated with a diminished
capacity of rat renal brush-border membranes in the kidney to reabsorb bile salts from the glomerular filtrate
(219). Mrp2 is also located at the apical membrane of the
proximal tubule (305, 306). However, in contrast to Isbt,
Mrp2 protein expression is upregulated on the apical
membrane of the rat renal proximal tubule (219) and is
Physiol Rev • VOL
5. Adaptive responses of bile salt transporters
in intestine
Studies on the effects of bile diversion and bile duct
obstruction on the pattern of expression of this transporter have been conflicting, with some showing downregulation (304) and others not (20). Isbt is not regulated
by the NHR FXR. However, bile salts do effect the expression of the ileal bile salt binding protein, I-Babp, a 17-kDa
protein that contains an FXR response element in its
promoter (118). FXR nullizygous mice do not upregulate
I-Babp when fed excessive amounts of cholic acid compared with the wild-type control animals (326). Because
I-Babp binds to the cytoplasmic domain of Isbt (199), it is
likely that bile salts can regulate the functional expression
of the bile salt transporter through effects on the expression of I-Babp (65, 300, 348). A recent study suggests that
MRP3 expression in enterocytes is induced by bile acids
via LRH-3/FTF (147).
6. Placenta
Maternal cholestasis (e.g., CBDL in pregnant rats)
impairs bile salt transfer from the fetus to the mother by
impairing bile salt transport at both the basolateral (fetalfacing) and apical (maternal-facing) membrane (232, 315).
The molecular basis remains to be clarified. Some of the
functional changes, however, appear to be related to reduction of trophoblast tissue (232).
IV. LIVER REGENERATION
Two-thirds partial hepatectomy of rat liver induces
liver regeneration and results in changes of bile salt trans-
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It is not clear if Isbt in the luminal membrane of
cholangiocytes has a significant function under normal
physiological conditions, but its expression is upregulated
when bile ducts proliferate in the bile duct obstructed rat
(219). Under these circumstances Isbt may function to
remove bile salts from the obstructed biliary lumen in
response to continued excretion of bile salts at the canalicular membrane of hepatocytes (Fig. 5). Recent studies
indicate that isolated large cholangiocytes from rat liver
are capable of sodium-dependent taurocholate uptake
and that during bile duct obstruction, Isbt expression is
also induced on the luminal membrane of small cholangiocytes by bile salts (9, 225). Isbt protein levels are also
increased in total liver homogenates after 2 wk of bile
duct ligation when cholangiocyte proliferation is pronounced (219). These findings suggest that bile duct proliferation may be an adaptive response to the retention of
toxic bile salts and feedback to facilitate bile salt clearance from the obstructed biliary lumen (25, 217, 219, 225).
Mrp3/MRP3 is also normally expressed at the basolateral membrane of rat and human cholangiocytes and
remains at this location as cholangiocytes proliferate during cholestasis induced by bile duct obstruction (137, 194,
331). Given the high affinity of Mrp3 for cholestatic bile
salts, it is likely that Mrp3/MRP3 plays the major role in
the return of bile salts from cholangiocytes to the systemic circulation when they are reabsorbed from the
biliary lumen (67, 113). MDR1 is also strongly expressed
in proliferating ductules from patients with PBC where it
might have a protective role (401). Although mutations
have not been identified in human MDR1 and bile salts are
not substrates for this transport protein, altered patterns
of expression of this transporter might reduce the capacity of the cholangiocyte to defend itself from toxic compounds. In such circumstances cholangiopathies might
develop.
associated with an increased ability to excrete the Mrp2
substrate para-aminohippurate (352) (Fig. 5). Conjugated
bilirubin, taurolithocholate sulfate, and bile all increase
Mrp2 expression in renal proximal tubular epithelial cells
(352). This adaptive response would facilitate the renal
excretion of divalent organic anions such as bile salt
sulfates and glucuronides that accumulate during cholestasis. Mutations in the Isbt gene in humans result in
primary bile salt malabsorption at the terminal ileum
(267), but the effect of these mutations on cholangiocyte
or renal handling of bile salts is not known. Nevertheless,
reciprocal differences in expression of Isbt and Mrp2
between cholangiocytes and kidney suggest that there
may be tissue-specific factors that regulate the expression
of these two bile salt transporters. Preliminary studies
indicate that cholestasis induced by CBDL in the rat results in the rapid loss of the NHRs, RXR-␣, FXR, and SHP
expression in the liver but not the kidney where renal
levels of FXR and SHP RNA and protein were increased
(82). Thus differences in transporter expression in liver
and kidney may be related to different patterns of NHR
responses to cholestasis in the two tissues.
BILE SALT TRANSPORTERS
porter expression (111, 368). At the basolateral membrane of hepatocytes, Ntcp mRNA and protein is markedly diminished. Oatp1 and -2 RNA levels decline while
Oatp1 protein expression falls and then recovers to normal levels while Oatp2 protein remains depressed. Mdr1
RNA expression is increased by partial hepatectomy. At
the canalicular membrane, Bsep RNA expression is variably expressed, but the protein remains unchanged. Mrp2
RNA and protein expression are either unchanged or
diminished. In contrast, Mdr1 and Mdr2 RNA levels are
increased. Functional correlates of these molecular responses to partial hepatectomy have not been determined.
Advances in the field of bile salt transport have
greatly accelerated since the advent of molecular cloning.
Classic biochemical and physiological studies, long the
traditional areas of study, have given way to an era of
functional genomics whereby the function and regulation
of the bile salt transporters are being understood at a
molecular level. In this review we have presented what is
currently known about the normal physiology of bile salt
transport in the liver and other extrahepatic tissues, discussed the rapidly expanding area of transcriptional and
posttranscriptional regulation of the bile salt transporters,
and examined the defects in bile salt transport that occur
in both genetic and acquired cholestatic disorders. As we
wrote this review, we were impressed with the rapid rate
of acceleration of publications in this field, making it
impossible to remain entirely current. We also apologize
to those whose work we overlooked or were unable to
include because of space. A number of recent reviews are
also cited in the text for the reader interested in following
one or more aspects of this field in greater detail.
Address for reprint requests and other correspondence:
J. L. Boyer, Yale University School of Medicine, PO Box 208019,
333 Cedar St., New Haven, CT 06520-8019 (E-mail: james.
[email protected]).
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