Hepatocellular Transport in Acquired Cholestasis: New Insights Into Functional, Regulatory and Therapeutic Aspects

Clinical Science (2008) 114, 567588 (Printed in Great Britain) doi:10.
1042/CS20070227
Hepatocellular transport in acquired

cholestasis: new insights into functional,
regulatory and therapeutic aspects
Marcelo G. ROMA, Fernando A. CROCENZI and Enrique A. SANCHEZ

POZZI
Instituto de Fisiologa Experimental (IFISE), Facultad de Ciencias Bioqumicas y Farmaceuticas (CONICETU.N.R.), Suipacha
570, S2002LRL, Rosario, Argentina
The recent overwhelming advances in molecular and cell biology have added enormously to our
understanding of the physiological processes involved in bile formation and, by extension, to
our comprehension of the consequences of their alteration in cholestatic hepatopathies. The
present review addresses in detail this new information by summarizing a number of recent
experimental findings on the structural, functional and regulatory aspects of hepatocellular
transporter function in acquired cholestasis. This comprises (i) a short overview of the
physiological mechanisms of bile secretion, including the nature of the transporters involved and
their role in bile formation; (ii) the changes induced by nuclear receptors and hepatocyte-enriched
transcription factors in the constitutive expression of hepatocellular transporters in cholestasis,
either explaining the primary biliary failure or resulting from a secondary adaptive response;
(iii) the post-transcriptional changes in transporter function and localization in cholestasis,
including a description of the subcellular structures putatively engaged in the endocytic
internalization of canalicular transporters and the involvement of signalling cascades in this effect;
and (iv) a discussion on how this new information has contributed to the understanding of the
mechanism by which anticholestatic agents exert their beneficial effects, or the manner in which
it has helped the design of new successful therapeutic approaches to cholestatic liver diseases.
INTRODUCTION
Cholestasis is a frequent, prominent and often severe
manifestation of many liver diseases. It can be defined as
an impairment of normal bile flow secondary to structural

or biochemical abnormalities of the liver and/or the biliary tree. These alterations may involve: (i) rapid changes
in transporter function, e.g. due to drug-induced direct
Key words: bile, cholestasis, hepatocellular transport, liver disease, nuclear receptor, therapeutic target.
Abbreviations: ABC transporter, ATP-binding-cassette transporter; AE2/Ae2, anion exchanger 2; AQP, aquaporin; ASBT/Asbt,
apical Na+ -dependent bile salt transporter; BSEP/Bsep, bile salt export pump; CAR, constitutively activated receptor; CYP/Cyp,
ctyochrome P450; E2 17G, oestradiol 17-d-glucuronide; EE, 17-ethynyloestradiol; ER, oestrogen receptor; ERK, extracellularsignal-regulated kinase; FXR, farnesoid X receptor; HNF, hepatocyte nuclear factor; IL, interleukin; JNK, c-Jun N-terminal kinase;
LPS, lipopolysaccharide; LRH1, liver receptor homologue 1; LXR, liver X receptor; MAPK, mitogen-activated protein
kinase; MRP/Mrp, multidrug resistance-associated protein; NHE, Na+ /H+ exchanger; NTCP/Ntcp, Na+ -taurocholate cotransporting polypeptide; OATP/Oatp, organic anion-transporting polypeptide; OST/Ost, organic solute transporter; PBC, primary
biliary cirrhosis; PFIC, progressive familial intrahepatic cholestasis; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C;
PPAR, peroxisome-proliferator-activated receptor ; PSC, primary sclerosing cholangitis; PXR, pregnane X receptor; RAR,
retinoic acid receptor ; RXR, retinoid X receptor ; SHP, small heterodimer partner; Stat5, signal transducer and activator of
transcription 5; SULT/Sult, sulfotransferase; t-BOOH, t-butylhydroperoxide; TLC, taurolithocholate; TNF, tumour necrosis
factor ; UDC, ursodeoxycholate; TUDC, tauroUDC; UGT/Ugt, UDP-glucuronosyltransferase; VDR, vitamin D receptor.
Correspondence: Dr. Marcelo G. Roma (email mroma@fbioyf.unr.edu.ar).

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M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi
inhibition of relevant transporters or changes in their

localization; and (ii) changes in transporter expression,
due to inhibition of carrier synthesis or exacerbated degradation. As a consequence of these primary cholestatic
alterations affecting primarily hepatocyte bile formation
(hepatocellular cholestasis), or in response to an intraor extra-hepatic blockage of the bile transit (obstructive
cholestasis), the liver develops a number of secondary
adaptive changes to minimize the detrimental effects of
toxic biliary compounds retained as a consequence of the
secretory failure. This adaptive response comprises,
among other modifications, changes in the expression
of both basolateral and canalicular transporters mediated
by the co-ordinated activation of a number of nuclear
hormone receptors. These regulators act as transcription
factors that directly interact with and control the expression of genomic DNA on binding a relevant ligand. These
ligands are, more commonly, retained bile salts or conjugated bilirubin, which function as sensors of secretory
failure.
The aim of the present review is to give an insight into
recent advances in the understanding of the mechanisms of canalicular transporter alterations in acquired
cholestasis, with particular emphasis in the structural and
functional alterations leading to transport dysfunction.
In addition, regulatory aspects involved in the adaptive
responses against the detrimental effects of the cholestatic
injury will be examined. Finally, we will discuss how
certain anticholestatic agents in current therapeutic use
may exert beneficial effects, and the mechanistic basis
of novel therapeutic strategies based on the information
obtained from these basic studies.
PHYSIOLOGICAL MECHANISMS OF BILE

FORMATION
Bile formation is an osmotic process driven by the
vectorial transport of solutes into bile. This induces
passive water movement from blood into bile in response
to osmotic gradients, via the water channels AQP
(aquaporin) 9 and AQP8 localized at the basolateral
and the apical membranes respectively [1,2]. For this to
occur, biliary solutes need to be actively concentrated and
retained into a confined space, i.e. the bile canaliculus.
Only bile salts, glutathione, either reduced (GSH) or
oxidized (GSSG) [3] and, perhaps, HCO3 [4] fulfil these
requirements. This primary canalicular secretion is modified further by cholangiocytes during its transit along
bile ducts. This process is a result of a balance between
hormone-dependent secretion of water and electrolytes, and the obligatory absorption of water, electrolytes
and organic solutes [5]. Figure 1 shows a schematic
representation of the main solutes and transporters
relevant to bile flow formation at both the hepatocellular
and the bile ductular levels.

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Bile salts are the predominant organic solutes in

bile and the main determinants of bile flow. They are
mainly taken up by the NTCP/Ntcp (Na+ -taurocholate
co-transporting polypeptide; SLC10A1/Slc10a1), driven
by the Na+ gradient maintained by the Na+ /K+ -ATPase
pump [6]. A remaining fraction is taken up by a
Na+ -independent transport system mediated by OATP/
Oatp (organic anion-transporting polypeptide) family of
transporters, which are non-electrogenic in nature [7].
In addition to conjugated and unconjugated bile salts,
OATPs/Oatps accept other amphipathic compounds,
such as bilirubin glucuronides and, maybe, unconjugated bilirubin. Four OATPs have been cloned
and characterized from human liver, namely
OATP1A2 (SLCO1A2/SLC21A3; formerly OATPA), OATP1B1 (SLC21A6; formerly OATP-C or
LST-1), OATP1B3 (SLC21A8; formerly OATP-8) and
OATP2B1 (SLC21A9; formerly OATP-B). All but the
latter are involved in bile-salt uptake, with OATP1A2
being the most important one [8]. For unconjugated
bilirubin [9] and bilirubin monoglucuronides [10], both
OATP1B1 and OATP1B3 have been proposed to be
involved. Although OATP1B1 was thought to have a
far higher efficiency for unconjugated bilirubin than
OATP1B3 [10], this finding was not supported by others
[11]. Bilirubin diglucuronides appear to be transported
only by OATP1B1 [10]. There are three Oatps identified
in rats, namely Oatp1a1 (Slc21a1; formerly Oatp1),
Oatp1a4 (Slc21a5; formerly Oatp2) and Oatp1b2
(Slc21a10; formerly Oatp4 or Lst-1). Oatp1b2 is the
rodent orthologue of both OATP1B1 and OATP1B3
[12]. All of them are involved in the uptake of conjugated
and unconjugated bile salts [8,13]. Oatp1a1 and Oatp1a4
are involved in bilirubin monoglucuronide transport
[13,14].
After traversing the cell by Ficks diffusion bound to
high-affinity cytosolic proteins, monoanionic bile salts
are excreted in the canalicular pole by BSEP/Bsep (bile
salt export pump; ABCB11), an ABC transporter (ATPbinding-cassette transporter) [15]. In contrast, canalicular
efflux of divalent, bipolar sulfated or glucuronidated bile
salts is mediated by MRP2/Mrp2 (multidrug resistanceassociated protein 2; ABCC2/Abcc2). This carrier is also
engaged in the biliary excretion of bilirubin mono- and
di-glucuronides [16].
Another key determinant of bile flow is glutathione,
both in its reduced (GSH; approx. 80 %) and in its
oxidized (GSSG) forms [3]. The liver is the main site of
glutathione synthesis, and biliary glutathione comes from
this intracellular source. A high-affinity electrogenic
canalicular carrier for GSH has been functionally characterized, but not cloned [17]. This transporter actively
exports GSH into bile, although it can transfer GSSG
with lower affinity. On the contrary, GSSG is excreted with higher affinity by Mrp2, which can also transfer
GSH, but with low affinity [18].
Hepatocellular transport in acquired cholestasis
Figure 1 Schematic representation of the main transport proteins involved in bile flow generation in both hepatocytes
and cholangiocytes
ATP-dependent transporters are depicted as brown/orange circles. For the sake of simplicity, only rodent transporters are displayed, but human orthologues of all of
these transporters have been identified. See the text for details on both rodent and human transporters. Br, bilirubin; BS , bile salts; Glc, glucose; Glu, glutamate;
GSH, glutathione; OA , organic anions; VIP, vasoactive intestinal peptide.
Another putative candidate to partially determine
bile flow is HCO3 [4]; however, it remains questionable whether bile-to-plasma HCO3 gradients can be
maintained, due to the high paracellular permeability of
this anion [19]. Overall, HCO3 transport involves the
sequential participation of three different transporters,
namely Na+ /K+ -ATPase, NHE (Na+ /H+ exchanger),
localized at the basolateral domain, and AE2/Ae2 (anion
exchanger 2; SLC4A2/slc4a2), localized at the apical
domain. Na+ /K+ -ATPase activity maintains plasma-tocytosol Na+ gradients for basolateral efflux of H+ via
NHE. The extruded H+ neutralizes HCO3 in plasma,
enabling its passive diffusion as CO2 . After conversion
of CO2 back into HCO3 by intracellular carbonic
anhydrase, the anion is counter-transported with Cl at
the canalicular domain by AE2/Ae2.
Canalicular bile flow is modified further during its
transit along the bile ducts by both secretory and absorptive processes [20]. Ductular fluid secretion is driven
by the secretin-regulated cAMP-dependent output of an

HCO3 -rich fluid secreted via the Cl /HCO3 exchange
system AE2/Ae2. Exchange is driven by the out-toin Cl concentration gradient, which is maintained by
the Cl efflux across the apical membrane via the
secretin-activated CFTR (cystic fibrosis transmembrane
regulator). As with secretin, VIP (vasoactive intestinal
peptide) [21,22] and bombesin [23,24] stimulate ductular
HCO3 -rich fluid both in rodents and humans. The
effects of these peptides is cAMP-independent, at least in
rodents [22]. Blood-to-bile water movement at the ductular level is facilitated by constitutive AQP4 in the basolateral membrane, and secretin-stimulated AQP1 in the
apical membrane [1,2]. On the other hand, the absorption
of ductular water and electrolytes is driven by osmotic
gradients created by bile-to-plasma transport of electrolytes and organic solutes. They comprise glutamate,
glucose, transported by SGLT1 and GLUT1 at the apical
and basolateral domains respectively, and bile salts, taken

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up by ASBT/Asbt (apical Na+ -dependent bile salt transporter; SLC10A2/slc10a2), followed by basolateral extrusion via the export pump MRP3/Mrp3 (ABCC3/Abcc3)
and the heterodimeric OSTOST/OstOst (where
OST/Ost is organic solute transporter) [5,25].
ALTERATIONS IN CANALICULAR
TRANSPORTERS IN ACQUIRED CHOLESTASIS
Changes in hepatobiliary transporter expression/activity
in acquired cholestasis have been reported to occur at the
transcriptional, post-transcriptional or post-translational
level. These pathomechanisms result in changes in
transporter activity in days, hours or minutes/seconds
respectively.
Changes in transporter expression in

cholestasis
Decreased expression of hepatobiliary transport systems
at the protein level can be the primary cause of the cholestatic injury or, alternatively, the secondary consequence of adaptive changes in transporter expression. In
the former case, a clear distinction between the mechanisms that actually cause the secretory failure from the
cascade of changes resulting from this initial cholestatic
insult is difficult to establish. These secondary adaptive
changes can be dissected better in experimental models of
obstructive cholestasis (bile duct ligation). In this case, the
primary cholestatic insult occurs at an extrahepatic level,
so that all of the hepatic changes can be regarded as adaptive. It should be stressed, however, that these animal
models cannot be extrapolated unequivocally to human
cholestatic conditions, due to differences in transcriptional regulation, transporter specificity and the length
of the cholestatic insult (acute in laboratory animal
compared with chronic in humans).
Adaptive changes in the expression of transporters and

metabolic systems in cholestasis
In several animal models of experimental cholestasis,
and in several human cholestatic liver diseases as well,
there is a common pattern of adaptive responses involving
changes in transporter expression. This response protects
the hepatocyte from the retention of toxic biliary
compounds, particularly bile salts and bilirubin [26]. The
adaptive changes comprise down-regulation of basolateral uptake systems, accompanied by the up-regulation
of basolateral extrusion systems (Figure 2). This greatly
helps to maintain intracellular concentrations of internalized metabolites, or de-novo-synthesized ones, at a safe
low level, despite ongoing secretory failure. This adaptive
transporter regulation is assisted further by repression
of the bile-salt-synthesizing enzymes Cyp7a1 and
Cyp8b1 (where Cyp is cytochrome P450), and the up
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regulation of phase I and phase II bile-acid-metabolizing

enzymes. Phase I bile-acid-hydroxylating enzymes
comprise Cyp3a11 (the rodent homologue of human
CYP3A4) and Cyp2b10 (the rodent homologue of
human CYP2B6). Phase II-conjugating enzymes are
Sult2a1 (where Sult is sulfotransferase) and Ugt2b4
(where Ugt is UDP-glucuronosyltransferase). Bile acid
hydroxylation (mainly at 1, 2, 4, 6, 6, 22 or 23
positions) results in an increased intracellular pool of
less toxic polyhydroxylated bile salts. Most of these
transformations facilitate the excretion of bile acids
into the bile (sulfation) or into the blood for subsequent urinary elimination (hydroxylation, sulfation and
glucuronidation) [27].
Ntcp [2831], Oatp1a1 [28,31,32] and Oatp1b2 [33]
protein levels are decreased in several animal models
of experimental cholestasis at a transcriptional level
[34]. This helps to maintain at a minimum the uptake
of potentially toxic compounds arriving to the liver,
such as the bile salts reabsorbed by the intestine and
bilirubin. On the other hand, Oatp1a4 is up-regulated in
bile-duct-ligated mice [33]. Organic substrate transport
carried out by Oatp1a4 is potentially bidirectional [35]
and, therefore, can reverse its transport direction in
conditions of bilirubin conjugate/bile acid overload, thus
acting as an alternative export system in cholestasis.
In addition to impairing uptake, the cholestatic
insult enhances routes for the basolateral extrusion of
organic anions. The basolateral membrane of rodent
cholestatic hepatocytes overexpresses several ATPdependent organic anion efflux pumps belonging to
the Mrp family, such as Mrp1 (ABCC1/Abcc1) [34,36,37],
Mrp3 (ABCC3/Abcc3) (in rat [28,3843], but not in
mouse [44]), Mrp4 (ABCC4/Abcc4) [34,44,45] and Mrp5
(ABCC5/Abcc5) [34,39]. In experimental obstructive
cholestasis in rodents, all of these increased expressions
occur at a protein level and are, at least in part,
transcriptional in nature [34]. These ATP-dependent
carriers extrude divalent bile acids (e.g. bile acid sulfoconjugates) and monovalent bile acids (glyco- and
tauro-conjugates) in rodents [13]. In humans, specificity
and transport mechanisms may vary. For example,
MRP3 transports only bile acid glycoconjugates with
low affinity [46,47], and MRP4 co-transports bile acids
with GSH [48]. In addition, the recently identified
OSTOST/OstOst, a heterodimeric transporter
that exports bile salts and their conjugates back to
plasma, is also up-regulated at the transcriptional level in
both the bile-duct-ligated mice and in cholestatic patients
with PBC (primary biliary cirrhosis) [49]. The relative
importance of each of these basolateral extrusion carriers
to transport biliary substrates, and their role in protecting
the hepatocyte against a cholestatic insult, may be
differential. Experiments in Mrp4/ mice with bile duct
ligation revealed that hepatic Mrp4 plays an important
role in the adaptive response to obstructive cholestatic
Figure 2 Adaptive changes in obstructive cholestasis in the expression of transporters and enzymes involved in bile salt
synthesis and bile salt/bilirubin conjugation in the liver, kidney and intestines
Note that, for simplicity, only changes in rodent gene products are displayed. Most of the adaptive changes at the hepatocellular level have been confirmed
in patients with cholestasis. On the contrary, most of the adaptive response in ductular epithelia, kidney and intestines described in rodents remains to be confirmed in
humans. For details, see the text. Br, unconjugated bilirubin; Br-G, bilirubin glucuronides; CFTR, cystic fibrosis transmembrane regulator; OA , organic anions; BS ,
bile salts.
liver injury by preferentially improving the extrusion of
bile salts into the blood. Indeed, more extensive damage
and lower serum bile salt levels occurred in Mrp4/
mice compared with the wild-type, despite both Mrp3
and OstOst being up-regulated [44]. Conversely,
bile-duct-ligated Mrp3/ mice had no differences
compared with the wild-type in both serum bile salt
levels and liver histology [43,50]; Mrp3/ mice had
lower serum levels of bilirubin, suggesting a preferential
role for Mrp3 in the basolateral extrusion of bilirubin
glucuronides.
The bile salt conjugates and the bilirubin glucuronides

that are not taken up by the hepatocyte, or that are
effluxed into the systemic circulation after uptake, can
be eliminated in urine. This alternative excretory route is
enhanced under cholestatic conditions [12]. The increased
efficiency of the kidney to detoxify these compounds
occurs by (i) increased passive glomerular filtration,
(ii) repressed transcription, leading to diminished protein
levels of Asbt [51], a transporter involved in tubular
re-absorption of bile salts, and (iii) the co-ordinated overexpression at the protein level of basolateral Oat1 [52]

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and of the apical transporter Mrp2 [38,51]; Mrp2 mRNA

levels did not increase, suggesting post-transcriptional
mechanisms [51]. Similarly, renal Mrp4 which, unlike
liver, has an apical localization in kidney, is up-regulated
transcriptionally in mouse kidney [53,54], although it is
down-regulated at the post-transcriptional level in the
rat [45].
The final hepatic fate of the cholephilic compounds
under physiological conditions is canalicular transfer,
which represents the rate-limiting step in the overall hepatic handling. In obstructive cholestasis, secondary inhibition of the expression of canalicular transporters occurs.
These changes can be regarded, however, as beneficial,
since this may help to protect bile ducts from mechanical
damage by an increase in biliary pressure [53,55] or from
cholangiocyte necro-apoptosis induced by toxic biliary
solutes [56]. Both Mrp2 protein expression and mRNA
levels were considerably impaired in obstructive cholestasis in rats [5759] and humans [60,61]. Bsep expression
is less altered by far [59,62]. An adaptive response against
this is the enhancement in the bile salt cholehepatic
shunt pathway. This mechanism removes bile salts
from the biliary space and maintains in operation
the hepato-cholangiocyte flux of bile salts, despite
ongoing bile duct obstruction. A key ductular bile salt
transporter involved in this response is Asbt. Although
the expression of this transporter is maintained per
cholangiocyte, the number of intrahepatic bile ducts
markedly increases in bile-duct-ligated rats (approx. 10fold after 1 week) [25]. Improvement in Asbt-mediated
bile acid transport through the cholangiocyte apical
membrane in bile-duct-ligated animals is assisted by a
concomitant overexpression of the basolateral bile acid
transporters Mrp3 [40] and OstOst [49]; Ost
Ost induction was only confirmed at the mRNA level.
Multiplication of bile ducts also induces a proportional
increase in the biliary tree death space. This helps to
substitute the intestines as the major reservoir of the
bile salt pool when bile salt enterohepatic recirculation is
impaired [63]. Down-regulation of intestinal Asbt protein
levels also occurs. This decreases the amount of bile salts
returning to the liver, thus reducing further the liver
bile salt load. Adaptive down-regulation of Asbt was
observed in bile-duct-ligated rats, the mechanism being
post-transcriptional in nature [64].
Commonly, the acquired changes in transporter expression in human cholestatic liver disease are consistent
with those occurring in experimental animal models of
cholestasis, although the mechanisms accounting for
these expression changes may vary. Generally, the contribution of transcriptional mechanisms is more limited,
or absent, in humans compared with rodents, with posttranscriptional/post-translational mechanisms being
more relevant [13,6567].
As in rodents, the down-regulation of basolateral uptake transport systems is apparent in chronic cholestatic

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diseases in humans. NTCP and OATP1B1 protein levels

decrease in inflammation-induced icteric cholestasis (e.g.
cholestatic alcoholic hepatitis) [65], late-stage PBC [66],
PFIC2 and PFIC3 (type 2 and 3 progressive familial
intrahepatic cholestasis respectively) [68], PSC (primary
sclerosing cholangitis) [67] and extrahepatic biliary atresia
[69]. OATP1B3 is also down-regulated in PFIC1 (type 1
PFIC) and PFIC2 at the protein level [68]. Similarly,
adaptive increments in protein levels of the hepatocellular basolateral export pumps MRP1 [70,71], MRP3
[66,70,71], MRP4 [71,72] and MRP5 [71] have been reported in late-stage PBC. Similarly, MRP3 [73] and MRP4
[68] protein levels were increased in PFIC3. Finally, increases in MRP3 have been reported in both biliary atresia
[73] and biliary obstruction, secondary to carcinomas
of the biliary tract or pancreas [61]. The mRNA levels of
the basolateral extrusion transporter OSTOST are
also augmented in the liver of patients with advanced
PBC [49]. Extensive up-regulation at the mRNA level of
OSTOST, but not of MRP3 and MRP4, may help
to explain why the induction of OSTs is more profound
than that of MRP3 and MRP4 in PBC [49]. OATP1B3,
which remains highly expressed in liver under cholestatic
conditions, behaves as a GSH-dependent symporter, and
has recently been proposed to be an additional route for
the basolateral extrusion of cholephilic organic anions,
including bile salts, in human cholestatic diseases [74].
At the canalicular pole, and similar to the findings
in bile-duct-ligated rats, MRP2 expression is decreased
at the protein level in cholestatic liver diseases, although
the changes are only observed in advanced stages of the
disease and are less dramatic in intensity. For example,
MRP2 protein expression only decreased in some, but
not all, patients with stage IV PBC [75] and remained unaffected in stages IIII PBC [66,75]. Similarly, MRP2 and
BSEP mRNA levels fell only in livers from poorly drained
patients with obstructive extrahepatic cholestasis, but
not in the well-drained ones [61]. Finally, BSEP protein
expression was relatively well preserved in patients with
extrahepatic biliary atresia, even in the untreated ones
[76].
In human cells, other than hepatocytes, the existence of
an adaptive response against cholestasis remains largely
speculative, although some indications are emerging.
Human cholangiocytes actively proliferate in response
to many cholestatic conditions, including obstructive
cholestasis and PBC [77]. MRP3 protein levels were
frequently increased in proliferative cholangiocytes from
livers of patients with various forms of cholestasis, as
revealed by immunohistochemical studies [70,73]. In
the intestine, adaptive down-regulation of ASBT in
patients with obstructive cholestasis has been reported
[78]. Finally, although all of the transporters involved in
the renal response to cholestasis have been identified in
human kidney, the occurrence of these adaptive changes
in patients with cholestasis remains to be confirmed.
Role of nuclear receptors and hepatocyte-enriched

transcription factors in adaptive response in cholestasis
Adaptive changes in expression of hepatocellular transporters are mediated mainly by the co-ordinated modulation of a number of transcription factors, including the
nuclear orphan receptors and liver-enriched HNFs (hepatocyte nuclear factors). Most of the information on these
mechanisms was obtained from rodent models of cholestasis, by using either specific ligands to activate nuclear
receptors or knockout animals with selective deficiency in
the synthesis of transcription factors. However, these regulations remain largely unconfirmed in human cholestatic
disease. It should be borne in mind that changes involving
these regulators are transcriptional in nature and, as stated
above, transcriptional mechanisms may be less relevant in
humans compared with rodents [13,6567].
The nuclear receptors thought to be involved in feedback or feedforward regulation of carrier genes in
cholestasis comprise, among others, FXR (farnesoid X
receptor; NR1H4), PXR (pregnane X receptor; NR1I2),
CAR (constitutively activated receptor; NR1I3), VDR
(vitamin D receptor; NR1I1), LXR (liver X receptor;
NR1H3), RAR (retinoic acid receptor ), LRH1 (liver
receptor homologue 1; NR5A2), PPAR (peroxisomeproliferator-activated receptor ; NR1C1) and SHP
(small heterodimer partner; NR0B2). All except the two
latter heterodimerize with RXR (retinoid X receptor
; NR2B1), enabling high-affinity DNA binding and
further activation of gene transcription [12]. The ligands
involved in this adaptive response are biliary constituents
retained by secretory failure, such as bilirubin [79] and
hydrophobic hepatotoxic bile salts (e.g. chenodeoxycholate, deoxycholate and lithocholate) [80,81].
In rodents, FXR-mediated induction of the nuclear
repressor SHP inhibits Ntcp expression in experimental
obstructive cholestasis [82,83], which surpasses the
RXR/RAR-mediated activation of the Ntcp promoter
[83]. The SHP effect may be, at least in part, mediated
by a multistep, regulatory cascade involving repression
of the hepatocyte-enriched transcription factor HNF4,
which suppresses its activating effect on HNF1 [also
known as TCF1 (transcription factor 1)], a potent Ntcp
activator [84]. The human SHP promoter region also
contains FXR-response elements [85]. The involvement
of SHP was, however, challenged by studies showing
that Ntcp is down-regulated in bile-duct-ligated SHP/
mice, suggesting that SHP is not essential for Ntcp
repression [86]. As bile salts can repress HNF4
(NR2A1) transcription genes, a direct bile salt inhibition
of this transcription factor can help to explain this finding
[87]. In humans, the relevance of this latter transcriptional
cascade is doubtful, as neither HNF4, HNF1 nor
RXR/RAR binds the Ntcp promoter, suggesting
species differences in NTCP/Ntcp gene regulation
[84].
A similar FXR-mediated repression of the SHP/

HNF4/HNF1 cascade has been proposed to be a
primary mediator of the transcriptional impairment of
Oatp1a1 and Oatp1b2 in rodent models of cholestasis
[13,87]. Interestingly, OATP1B1, the gene of the human
orthologue of Oatp1b2, contains an HNF1-binding
site in its promoter region [88], suggesting that this latter
mechanism may be operative in patients with cholestasis
[89]. In addition, PXR and CAR up-regulate the Na+ independent bile-acid uptake transporter Oatp1a4 [33].
CAR is one of the nuclear receptors involved in the upregulation of Mrp3 and Mrp4 in rodents, thus enhancing
basolateral export pathways in these animal models [90
92]. Mrp3, but not Mrp4, overexpression is assisted by
PXR [90,93] and LRH1 [94]. In this latter case, another
factor mediating bile-acid-dependent induction would be
required, as bile acids are not LRH1 ligands. In contrast,
induction of Mrp3 and Mrp4 are FXR-independent
[53,95,96]. Furthermore, FXR appears to inhibit Mrp4
in rodent cholestatic liver, but CAR overrides this inhibitory effect [97]. In humans, the mechanisms of MRP3
induction may be different to rodents, as a CAR
cis-binding element could not be identified in the human
MRP3 promoter [98]. PXR-mediated up-regulation of
human MRP3 is an alternative possibility, as ligand-activated PXR has also been shown to induce MRP3/Mrp3
expression in human hepatoma cell lines [93]. LRH1induced MRP3 expression is also possible, as the MRP3
promoter contains a binding element for LRH1 [99].
Finally, MRP3 overexpression may also involve a
decrease in the expression of RXR/RAR in human
cholestasis. This nuclear receptor down-regulates MRP3
expression by abrogating the activity of the transcription
factor Sp1 on the MRP3 gene promoter [98].
FXR activates the basolateral bile-salt-exporting carrier OSTOST/OstOst, both in healthy rodents
[96] and in cholestatic rodents and humans [49]. Similarly, FXR transactivates OATP1B3, but not other OATP
isoforms [100]. Differential activation of OATP1B3 may
be related to its bile-salt-exporting function at the
basolateral domain [74].
In the canalicular pole, bile-salt-activated induction
of FXR is responsible for the maintenance of Bsep
expression under these cholestatic conditions by upregulating the otherwise decreased Bsep expression. This
regulatory role for FXR has been only confirmed in a
rodent model of obstructive cholestasis [53]; however,
the BSEP (ABCB11) gene also contains an IR1 (inverted
repeat 1) element in its promoter regions that directly
binds to the FXR/RXR heterodimer upon activation
by bile acids, resulting in enhanced transactivation of the
BSEP gene [101103].
Unlike Bsep, Mrp2 expression is extensively repressed
in cholestatic liver [57]. Obstructive cholestasis is
accompanied by systemic release of pro-inflammatory
cytokines [104]. The decrease in Mrp2 content in this

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condition appears to be due to a cytokine [IL (interleukin)

1]-dependent impairment in RAR expression
[105,106]. Cytokines also induce re-localization of the
nuclear receptor partner RXR from the nucleus to
cytosol, a finding shown in LPS (lipopolysaccharide)induced cholestasis [107]. If the latter event also occurs in
obstructive cholestasis, this may contribute further to the
reduced occurrence of RAR/RXR heterodimerization
and that of other nuclear receptors that transactivate
MRP2/Mrp2, such as FXR, PXR and CAR [108].
Nuclear receptors also mediate adaptive changes
in enzymes involved in bile salt synthesis and bile
salt/bilirubin detoxification [27,109,110]. Most of these
regulations have been observed in rodent models of
cholestasis, and the involvement of these regulatory
mechanisms in patients with cholestasis has been
inferred, but not determined, from studies in patients
without cholestasis receiving specific nuclear receptor
ligands for therapy or from studies in human cells lines.
FXR (via the SHP-mediated repression of LHR1 [85])
and PXR [80,111] inhibit de novo bile salt synthesis
by repressing the transcription of the genes encoding
Cyp7a1, the rate-limiting enzyme in the classic bile salt
biosynthetic pathway. In primary human hepatocytes,
activation of PXR by rifampicin promotes PXR interaction with HNF4 and further CYP7A1 inhibition
[112]. Cyp8b1, which controls the dihydroxy-totrihydroxy bile acid synthesis ratio, was also repressed,
thus regulating the bile salt hydrophobicity index. A
similar down-regulation of both Cyp7a1 and Cyp8b1
occurs after PPAR activation in rodents [113,114].
PXR and CAR enhance the expression of Cyp3a-mediated bile salt hydroxylation in rodents [80,92,111,115],
and this also appears to apply to humans. Indeed, a
response element in the human CYP3A promoter has
been identified for PXR [116] and CAR [117], and ligands
of both nuclear receptors stimulate CYP3A expression in
human hepatocytes [118]. Interestingly, administration
of the PXR ligand rifampicin stimulates CYP3A4
expression in otherwise healthy patients with cholesterol
gallstones [119]. HNF4 appears to be critically involved
in PXR- and CAR-mediated transcriptional activation
of CYP3A in humans. A specific cis-acting element in the
CYP3A4 gene enhancer has been identified that confers
HNF4 binding, thus permitting PXR- and CARmediated gene activation [120]. Finally, a role for LXR
in CYP3A4 induction has been proposed recently [121].
Rodent Ugt1a1 (UDP-glucuronosyltransferase 1A1)mediated bilirubin glucuronoconjugation is also activated
by PXR and CAR [90,92]. The PXR activator rifampicin
[122] and the CAR activator phenobarbital [123] both
induced human UGT1A1 protein expression.
CAR [90], and to a lesser extent FXR [124], PXR
[125,126], VDR [127] and LXR [128], activate SULT2A1/
Sult2a1. This enzyme enhances bile acid sulfation and
further renal excretion of the sulfated bile salts. Finally,

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both FXR [129] and PPAR [130] increase bile acid

glucuronidation by transactivating human UGT2B4.
Changes in transporter expression as a primary cause of

cholestasis
Changes in transporter expression in acquired hepatocellular cholestasis may represent the primary causal
factor of cholestatic dysfunction. Although adaptive
changes similar to those occurring in obstructive
cholestasis can also take place to protect the hepatocyte
against secondary manifestations of the cholestatic insult,
these adaptive changes may further aggravate the primary
secretory failure by reversing hepatocellular polarity.
Many acquired non-obstructive cholestatic disorders
involve primary changes in hepatocellular transporter
expression (hepatocellular cholestasis). They can be
divided in two large subgroups, namely (i) inflammationinduced cholestasis, which have cytokines as the common
deleterious effector, and (ii) pure (bland) cholestasis,
which is caused by drugs or hormones. It should,
however, be borne in mind that cholestatic diseases with
features of both obstructive and hepatocellular cholestasis
also exist. For example, PBC [75] and PSC [67], two
prototypical chronic cholestatic liver disease in humans,
develop bile duct obstruction by cholangiodestruction,
but inflammation-induced release of cytokines is also critically involved in the progression of the disease [131,132].
Inflammation-induced cholestasis
Pro-inflammatory cytokines produced as a consequence
of either infectious or non-infectious diseases are potent
inducers of intrahepatic cholestasis by acting as potent inhibitors of transporter expression [133]. Among the multiple pathologies in which inflammation-induced cholestasis is involved, sepsis-induced cholestasis is the prototypical one [134,135]. The cholestatic complication of
this disease has been associated with the action of the bacterial wall component LPS, and the consequent hepatic
release of the inflammatory cytokines IL1, TNF (tumour necrosis factor ) and IL6 [136]. Therefore LPS administration to rodents has been widely used as an experimental model to characterize sepsis-induced cholestasis.
This model was also helpful in understanding the cholestatic mechanisms of cytokines. This information is relevant to human cholestatic diseases other than sepsis, as cytokines are critical mediators of alcoholic, viral, autoimmune and drug-induced cholestatic hepatitis [133].
Protein expression of both Bsep and Mrp2 are
impaired in experimental sepsis in rodents [37,137,138],
although the alteration of Bsep appears to be less pronounced [62]. In rats, Mrp2, but not Bsep, expression
is modulated transcriptionally [37,137], whereas, in
humans, down-regulation of both BSEP and MRP2 is
post-transcriptional in nature, indicating marked species
differences [137].
The decrease in canalicular transporter expression

in LPS-induced cholestasis in rats is accompanied by
transcriptional down-regulation of Ntcp [139,140] and
several Oatps [136,139,140]. Up-regulation of Mrp1
and Mrp3 was also reported [37,140], although the upregulation of Mrp3 was not observed by others [136,141].
At least part of these alterations occur in human liver
slices exposed to LPS, in particular those involving
NTCP transcriptional down-regulation [137]. This
event correlated inversely with TNF and IL1 levels
[137].
Down-regulation of Mrp2, Ntcp and Oatps in
rodents may be due, in part, to repressed transcriptional
expression of both RXR/RAR and HNF1 [142],
which are transactivators of these transporters. LPSinduced cytokine release may play a key role [141,142].
Indeed, depletion of Kuppfer cells, the main hepatic
source of cytokines, counteracted the decrease induced
by LPS in the binding activity of the transcription
factors RXR/RAR and HNF1, and the associated
decrease in Ntcp expression in rats [143]. Cytokines
released after LPS administration to rodents also reduce
the binding activity of RXR/RAR by inducing a
shift in RXR from the nucleus into the cytosol, which
induces the removal of the heterodimeric partner from
the nucleus [107]. Phosphorylation of RXR by JNK
(c-Jun N-terminal kinase) after LPS-induced stimulation
of Kupffer cells [144] has been proposed to trigger
this re-localization [107]. JNK activation has been also
proposed to be a key determinant in the decreased
binding activity of HNF1. This effect appears to be
secondary to a decrease in HNF4 promoter activity
and protein expression, with JNK-mediated HNF4
phosphorylation having a critical role [145].
Selective inhibition of different cytokines in LPSinduced cholestasis in vivo revealed that IL1 is the
major cytokine involved in these effects [146], whereas
IL6 only plays a crucial role during the later stages of
endotoxaemia [147]. However, mechanisms independent
of cytokines, and not involving the repression of RXR/
RAR and HNF1, may also occur [146]. For example,
LPS-induced PXR activation has been shown to
contribute to Mrp2 down-regulation in mice [148].
Alterations in hepatobiliary transport systems
have been reported in inflammation-induced acute
icteric cholestasis caused by cholestatic alcoholic hepatitis, cholestatic autoimmune hepatitis and drug-induced
hepatitis [65]. A decreased expression of basolateral
uptake transporters (NTCP and OATP2) and canalicular
export pumps (BSEP and MRP2) was reported. This
was accompanied by low mRNA levels of NTCP and
OATP2 and, to a lesser extent, of BSEP. In contrast,
MRP2 down-regulation appears to occur by post-transcriptional mechanisms. Finally, MRP3 expression is
maintained, which may protect hepatocytes from further
accumulation of potentially toxic biliary constituents.
Pure (bland) cholestasis

Causes of pure (bland) cholestasis include hormones
and hormone derivatives, such as oestrogens, C17 -substituted testosterone and anabolic steroids, as well as a large
number of cholestatic drugs [149]. Among the several
cholestatic pathologies belonging to the latter group,
oestrogen-induced cholestasis in rats and its clinical
correlate, obstetric cholestasis, were studied with more
detail in terms of hepatocellular transporter expression
and these will be focused upon below.
Oestrogens have been proposed to be a key causal
factor in intrahepatic cholestasis in susceptible women
during pregnancy [150], or following administration of
oral contraceptives and postmenopausal replacement
therapy [151]. Owing to this relevant clinical correlate,
cholestasis induced by oestrogens in rodents is a common
experimental model of hepatocellular cholestasis. The
cholestatic injury is produced either over the course
of a few days by administration of EE (17-ethynyloestradiol), a model oestrogen, or within minutes by
E2 17G (oestradiol 17-d-glucuronide), an oestrogen
metabolite.
In the EE-induced cholestatic model, Bsep expression
is impaired slightly [62,152], whereas Mrp2 expression is
decreased to a higher degree at the post-transcriptional
level [57]. EE also induces transcriptional down-regulation of Ntcp [32,152,153] and Oatps [32,153,154],
together with the up-regulation of Mrp3 [42,58,154]. The
nuclear receptor ER (oestrogen receptor ) has been
recently proposed to mediate, at least in part, EE-induced
cholestasis and the changes in transporter expression
induced by the cholestatic agent [155]. Indeed, ER /
mice lack the EE-induced repression of the mRNA
expression of Ntcp, Oatp1a1, Oatp1a4, Bsep and bileacid-biosynthesis enzymes (Cyp7a1, Cyp7b1 and
Cyp8b1) [155]. On the contrary, other nuclear receptors,
such us ER, FXR, SHP, PXR and CAR, appear not to be
involved [155]. However, a role for FXR or SHP in other
species cannot be ruled out. Unlike this study in mice, EE
down-regulates FXR and up-regulates SHP, via ER, in
rats [156,157]. In addition, an oestrogen-induced decrease
in nuclear binding activity of Ntcp transactivators, such as
HNF1, C/EBP (CAAT/enhancer binding protein) and
PXR, has also been proposed to play a role in the downregulation of Ntcp [153]. Finally, an indirect mechanism
by which oestrogens influence the secretion of pituitary
hormones which, in turn, modify liver gene expression,
has been proposed recently [157]. In summary, EE
induces changes in bile acid metabolism and transport
through: (i) an interaction with nuclear receptors,
such as ER, FXR and SHP, (ii) a indirect interaction
with pituitary (or pituitary-dependent) hormones, and
(iii) as yet unidentified nuclear-receptor- and pituitary-independent mechanisms.
Although oestrogens are thought to be key causal
factors in obstetric cholestasis [150], the extent to which

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experimental EE-induced cholestasis in rats resembles

obstetric cholestasis during pregnancy is unknown.
Differences can be anticipated, however, since women
who develop obstetric cholestasis appear to have a
genetically linked susceptibility to the disease, and are
exposed to high levels of other hormones apart from
oestrogens (e.g., progesterone, prolactin and placental
lactogens) [13,150]. Owing to this complexity, animal
models of pregnancy-induced cholestasis are currently
unavailable, but the pattern of expression of transporters
in normal pregnant rats may provide some clues. As in
EE-induced cholestasis, transcriptional down-regulation
of Ntcp was apparent in pregnant rats, a finding
attributed to the down-regulation of HNF1 and
decreased promoter binding of RXR/RAR [158]. This
phenomenon was, however, not observed by others [159].
The limited, if any, decrease in Ntcp expression might
be attributed to the simultaneous stimulation of Ntcp
transcription by prolactin, an effect involving the binding
of Stat5 (signal transducer and activator of transcription 5)
to the Ntcp promoter [13,13,160]. Unlike in EE-induced
cholestasis, in which all Oatps were moderately downregulated, Oatp1a1 remains unchanged in pregnant rats,
whereas Oatp1a4 is only marginally decreased at both
the protein and mRNA levels [159]. Although a counterstimulatory effect of prolactin was not confirmed
for Oatp1a1 and Oatp1a4, the hormone up-regulates
Oatp1b2 expression via the Stat5 signal transduction
pathway [161]. The expression of basolateral export
pumps in pregnant rats also differs from that in
EE-induced cholestasis. Expression of both Mrp1
and Mrp3 is decreased at a post-transcriptional and
transcriptional level respectively [162]. Mrp6 mRNA
levels are also decreased [162]. Finally, at the canalicular
pole, Mrp2 expression was extensively reduced at a
post-transcriptional level, whereas Bsep expression is
not impaired. Bsep expression may be sustained by the
selective counter-stimulatory effect of prolactin on Bsep
expression [159]. Although moderate when compared
with the changes occurring in a pure oestrogen
model of cholestasis, the alterations in transporter
expression observed in pregnant rats suggest that
pregnancy represents an oestrogen-linked cholestaticprone condition that, when overlapping with subclinical
genetically linked alterations in bile formation in
susceptible women, may lead to overt cholestasis.
Manipulation of the expression of hepatocellular transporters

as a therapeutic strategy in cholestasis
The identification of a critical role for nuclear receptors
as mediators of the adaptive changes in hepatocellular
transporter expression in cholestasis has raised expectation about the employment of specific optimized ligands
of these nuclear receptors for the treatment of cholestatic
disorders [163165]. These new therapeutic approaches
are aimed not only at restoring the normal plasma-to
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bile excretory route by stimulating defective transporter

expression, but also at enabling alternative elimination
routes. This knowledge has also provided new rationales
for the understanding of the beneficial effects of wellestablished anticholestatic agents, which have been used
in an empirical manner in the past.
Among the anticholestatic agents, UDC (ursodeoxycholate) is the prototypical one. Feeding UDC to
normal rodents stimulated hepatocellular expression of
both Mrp2 and Bsep by transcriptional and post-transcriptional mechanisms respectively [166]. Expression at
both mRNA and protein levels of both of the basolateral
efflux pumps Mrp3 [167] and Mrp4 [96] was increased,
whereas that of Oatp1a1 was decreased (Figure 3). On the
other hand, expression of the uptake transporter Ntcp
[166] and the basolateral export transporter OstOst
[96] remained unaffected. Feeding UDC also stimulates
the protein expression of Mrp2 [167] and Mrp4 [96]
efflux pumps in mice kidney, and that of Mrp2 [167],
Mrp3 [167] and Mrp4 [96] in mouse intestines. Intestinal
OstOst mRNA was also elevated [96]. Finally, the
induction of bile-salt-hydroxylating enzymes [95] and
repression of bile-salt-synthesizing enzymes [96] also
occurs after UDC administration. Taken together, this
response enhances the overall capacity of the body to
detoxify toxic biliary constituents.
The effects of UDC in rodents were only partially
reproduced in humans. When administered to otherwise
healthy patients with cholesterol gallstones, UDC stimulated post-transcriptionally the expression of BSEP
and MRP4, but not MRP2, MRP3, OATP1B1 or
bile-salt-metabolizing enzymes in liver [168]. However,
the situation in patients with cholestasis may be far
more complex. For example, a positive correlation was
observed between the hepatic enrichment of UDC in
liver of UDC-treated patients with PBC and the hepatic
levels of OATP1B1 and MRP2, whereas BSEP levels had
no correlation [66]. This suggests that, unlike healthy
subjects, patients with PBC lack UDC-stimulatory
mechanisms of BSEP overexpression. Furthermore,
UDC would improve MRP2 or OATP1B1 levels only
when their expression is impaired by the disease.
Some of the effects induced by UDC in rodents appear
to involve the activation of nuclear factors. With the exception of the induction of Bsep by UDC, which appears
to be FXR-dependent in mice [167], the remaining responses to UDC are FXR-independent and may involve,
in part, other nuclear factors, probably PXR [95]. As most
of these effects occur spontaneously in the cholestatic
liver, UDC administration to bile-duct-ligated mice only
have a limited protective response [13]. More specific and
effective therapies using newly synthesized ligands that
bind FXR with higher affinity, e.g. GW4064 and 6-ethyl
chenodeoxycholic acid, are being actively tested; they
might counteract the reduced expression and activity of
FXR in cholestasis, particularly in cholestasis induced by
Figure 3 Beneficial effects of the anticholestatic therapeutic agent UDC on the expression of transporters and enzymes
involved in bile salt synthesis, and bile salt and bilirubin metabolism in the liver, kidney and intestines in normal rodents
At the hepatocellular level, UDC stimulates the expression of the canalicular transporters Bsep and Mrp2 and the basolateral efflux pumps Mrp3 and Mrp4. Expression
of Ntcp, Oatp1 and OstOst remain unaffected. The increased expression of Bsep has been attributed to UDC-induced activation of FXR, with the remaining
responses presumably involving other nuclear factors, such as PXR. UDC also induces beneficial changes in bile salt metabolism, particularly the repression of bile
acid synthesis from cholesterol and the stimulation of phase I bile acid metabolism, leading to more hydroxylated, less toxic, bile salts, which can be more easily
sulfated/glucuronidated and exported further via urine. This latter process is also stimulated by UDC by up-regulating both Mrp2 and Mrp4 efflux pumps in the kidney.
Finally, UDC up-regulates Mrp2, Mrp3 and Mrp4 efflux pumps in the intestine. Overexpression of the latter two efflux pumps contributes to preserve enterohepatic
bile salt recirculation. Note that the Figure shows the effects of UDC on healthy rodents. The extent to which these mechanisms are in operation in patients with
cholestasis is uncertain. See the text for further details.
pro-inflammatory cytokines (e.g. endotoxaemia) [169],
pregnancy [170] or oestrogens [156,157]. For example,
the FXR agonist 6-ethyl chenodeoxycholic acid has
beneficial effects in oestrogen-induced cholestasis [154],
a pathology that occurs with a decrease in FXR mRNA
levels [157]. This FXR ligand increases the expression of
Mrp2 and Bsep in EE-induced cholestasis and reduces
the transcriptional expression of Cyp7a1, Cyp8b1 and
Ntcp [154]. Although FXR-mediated stimulation of Bsep

expression can be beneficial in hepatocellular cholestasis
with impaired Bsep transport, it can be detrimental in obstructive cholestasis, where pressure in the obstructed bile
ducts can build up to hazardous levels. This explains why
FXR/ mice are more resilient to obstructive cholestasis
[97] and that UDC induces the FXR-mediated harmful
effects in bile-duct-ligated animals [55]. As most of the

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relevant acquired cholestasis in humans, such as PBC

and PSC, develops an obstructive/cholangiodestructive
component, this factor may narrow the therapy window
for treatments with FXR ligands [171].
Apart from improving the expression of key transporters in certain cholestatic diseases, in others UDC
largely enhances the activity of transporters without
affecting expression. For example, EE impairs Mrp2
expression and the biliary excretion of Mrp2 substrates,
but UDC co-administration largely restored this alteration without counteracting the Mrp2 down-regulation
[172]. The same holds true for the anticholestatic
hepatoprotector silymarin [173]. This might involve a
direct positive interaction of the anticholestatic agents
either with a putative regulatory site of the transporter
or with its lipid microenvironment.
The capability of UDC to induce beneficial changes in
bile acid metabolism in rodents, particularly repression
of Cyp7a1 and stimulation of Cyp3a11 (the rodent
homologue of human CYP3A4), has proved to be
limited in humans, but can be complemented with
simultaneous administration of PXR agonists, such as
rifampicin [119]. Rifampicin, which has also been used
for a long time in the treatment of cholestatic diseases,
preferentially stimulated beneficial changes in bile acid
metabolism [119]. This drug also enhances bilirubin-conjugating capability (via UGT1A1), bilirubin excretion (via
MRP2) and bile acid/bilirubin glucuronide retrograde
exportation (via MRP3) [119]. It should be highlighted
again that these stimulatory effects have been tested in
patients without cholestasis, but these mechanism may be
different in patients with cholestasis. If confirmed under
cholestatic conditions, similar complementary effects
of UDC with CAR agonists, such as phenobarbital or
6,7-dimethylesculetin (the main active component of a
herbal concoction used in traditional YinChin medicine
to treat neonatal jaundice) are to be expected, as CAR
co-ordinately regulates a similar group of detoxifying
genes to those induced by PXR (see above). In line with
this, FXR, PXR and CAR agonists protected against
hepatic bile acid toxicity in a complementary manner in
rats [92,115]. This preliminary encouraging information
supports the implementation of clinical trials using combinations of specific ligands targeting multiple but
complementary nuclear receptors.
Post-translational alterations of
hepatobiliary transporters
Bile flow can be impaired by certain cholestatic agents
within hours or even minutes, a time period incompatible
with changes in transporter expression at the protein
level. These acute cholestatic changes account, for
example, for the rapid cholestatic effects induced by drugs
in patients [174]. Moreover, certain chronic cholestatic
conditions do not involve changes in transporter expressions of a suitable magnitude to explain the extent of bile

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flow impairment, and anticholestatic agents may have beneficial effects without improving transporter expression.
In these cases, alterations in transporter intrinsic activity
or dynamic localization may play a crucial role [175].
Transporter inhibition by cholestatic drugs

A number of cholestatic agents cis-inhibit ATP-dependent Bsep-mediated bile salt transport in isolated rat
liver canalicular membrane vesicles, mostly in a competitive manner [174]. These agents include cyclosporin
A [176], glibenclamide [176], rifamycin [176], rifampicin [176], bosentan [177,178] and thiazolidinedione derivatives [179,180]. This effect was confirmed for human
BSEP with some of these compounds [178,181].
Flutamide was also shown to cis-inhibit BSEP [182].
On the other hand, some cholestatic compounds may
induce trans-inhibition of BSEP/Bsep. For example,
E2 17G has been suggested to trans-inhibit Bsep in rat
liver, as it requires an intact Mrp2-mediated E2 17Gtransporting activity to exert its cholestatic effect
[176,183]. This has been confirmed further in Xenopus
laevis oocytes co-expressing rat Bsep, the inhibition
mechanism being non-competitive in nature [183].
However, the relevance of this mechanism in the intact
liver is doubtful, as experimental manoeuvres that
increase the biliary E2 17G concentration attenuate
E2 17G-induced cholestasis [184,185], and others that
dilute the cholestatic compound in bile are not protective
at all [186]. The dependency of E2 17G-induced
cholestasis on intact Mrp2 activity, which indeed occurs
in vivo [187], can be explained alternatively by the Mrp2
inhibitory modulation of Bsep transport activity via, for
example, proteinprotein interactions.
PM4-S
(5-pregnan-3-ol-20-one
sulfate),
a
progesterone metabolite that accumulates in the serum of
women with pregnancy-induced cholestasis, has also
been shown to trans-inhibit rat Bsep in X. laevis oocytes
co-expressing rat Bsep in a non-competitive manner
[183]. This suggests that this metabolite impairs bile
flow in the isolated perfused rat liver, while being
excreted at concentrations higher than the K i for Bsep
trans-inhibition [183].
Changes in transporter localization

Impairment of transporter activity in cholestasis can also
occur by endocytic internalization of relevant canalicular
transporters, which re-localize into intracellular vesicular
structures, presumably the subapical endosomal compartment. If maintained with time in chronic cholestatic
conditions, this may lead to delivery of the protein to the
lysosomal compartment, followed by degradation [188].
This may contribute to the decreased post-transcriptional
expression of canalicular transporters shown to occur in
different cholestatic diseases.
Endocytic internalization of the canalicular transporters Mrp2 and Bsep occurs in several models of
experimental cholestasis, including bile duct ligation

[188], hyperosmotic perfusion [189,190] and oxidative
challenge [191193]. Administration of cholestatic
compounds, such as LPS [190], E2 17G [194,195], TLC
(taurolithocholate) [196,197] and cyclosporine A [198],
also induces this effect. In addition, Mrp2 internalization
has been reported to occur in post-cold ischaemic
rat liver, a phenomenon dependent on Kupffer cell
activation, with thromboxane A2 being the most likely
mediator [199]. Together with endocytic internalization,
abnormal expression of this canalicular transporter in
the apical end of the lateral membrane was also shown to
occur under sustained E2 17G-induced cholestasis. This
was attributed to mis-sorting of endocytosed transportercontaining vesicles localized at the pericanalicular area
to the closer lateral membrane domain [200].
Changes in localization of canalicular export pumps
have been also shown to occur in human cholestatic
hepatopathies, including PBC [201], obstructive
extrahepatic cholestasis [61,202], PSC [202], autoimmune
hepatitis [202] and acute intrahepatic cholestasis induced
by drugs, such as antibiotics, tiopronin, chlorpromazine,
NSAIDs (non-steroidal anti-inflammatory drugs)
and antidepressants [202204]. In the latter case,
redistribution of Mrp2 occurs towards the basolateral
membrane rather than into intracellular vesicles [204].
The mechanism by which endocytosis of canalicular
transporters occurs remains largely unknown, although
it is known that the process is microtubule-independent
[205]. Alterations of actin cytoskeletal integrity onset
transporter endocytosis [206], although retrieval of
canalicular transporters can also occur with intact
actin organization [195,197]. Proteins involved in the
cross-linking of actin filaments with plasma membrane
proteins, such as the ERM (ezrin/radixin/moesin) family
of proteins, may be altered in these cases, without any
apparent impairment of actin organization [207,208].
Interestingly, a deficient localization of radixin at the
canalicular membrane occurs in patients with PBC, which
is associated with retrieval of MRP2, MRP1, MDR3
and BSEP, together with co-localization of these carriers
with radixin in intracellular vesicles [201]. This finding
was confirmed for MRP2 in other human cholestatic
diseases, such as obstructive jaundice, PSC, autoimmune
hepatitis and drug-induced liver injury [202].
Accumulating evidence indicates that changes in canalicular transporter localization occurring in cholestasis
depend upon activation of critical signalling pathways
(Figure 4). One of the pathways involves classical Ca2+ dependent PKC (protein kinase C) isoforms (mainly
PKC in hepatocytes). Selective activation of Ca2+ dependent PKCs induces cholestasis and retrieval of Bsep
from the canalicular membrane and cholestasis in the
isolated perfused rat liver [209]. PKC activation also
induces redistribution of Mrp2 from the canalicular to
the basolateral membrane in HepG2 cells [210]. A critical
participation of Ca2+ -dependent PKCs in the retrieval of

Bsep and the associated bile salt secretory failure in tBOOH (t-butylhydroperoxide)-induced oxidative stress
has been reported recently by our group [193]. However,
the kind of canalicular protein that is internalized under
oxidative stress conditions and the signalling molecule(s)
involved appear to depend on the pro-oxidant agent employed. For example, the oxidizing compound ethacrynic
acid, which does not translocate classical but rather
novel PKC isoforms, internalizes Mrp2 selectively
without affecting Bsep [192]. The novel PKC isoform
PKC is also activated in TLC-induced cholestasis and
has been suggested to be involved in TLC-induced
cholestatic effect [211]. This phenomenon occurs in a
PI3K (phosphoinositide 3-kinase)-dependent manner, as
products of PI3K are potent activators of PKC [212].
Experimental therapeutic approaches

based upon the modulation of
post-translational alterations of
transporters in cholestasis
Direct transporter inhibition by cholestatic drugs is, in
most cases, reversed by withdrawing the drug, due to
the reversible nature of the phenomenon [213]. Similarly,
mechanisms involving changes in transporter localization
can be spontaneously reversed if the cholestatic insult is
transient. For example, canalicular transporters that are
internalized by a rapid bolus administration of E2 17G
are spontaneously re-targeted to the canalicular membrane by a microtubule-dependent mechanism within
2 h [214]. Some experimental therapeutic approaches
have been designed to prevent transporter internalization
and/or accelerate this re-insertion in order to avoid
irreversible consequences of sustained internalization, as
outlined below.
cAMP
cAMP is a second messenger that stimulates hepatocellular exocytic pathways [215] and partially prevents
the impairment of bile flow and internalization of ABC
transporters in E2 17G- and TLC-induced cholestasis.
Reduction in bile flow and activity of Bsep [195] and
Mrp2 [194] in the acute phase of E2 17G-induced cholestasis is partially prevented by the membrane-permeant
cAMP analogue dibutyryl cAMP. More significantly,
dibutyryl cAMP shortened the spontaneous recovery to
normal bile flow, Mrp2 function and Mrp2 localization
[194]. A similar acceleration of the re-insertion of endocytosed transporters has also been described by us for
Bsep in TLC-induced cholestasis [197]. In the IRHC
(isolated rat hepatocyte couplet) model, a preventive
effect of dibutyryl cAMP was observed on both E2 17G[195,216] and TLC- [197,216] induced Bsep relocation.
In this case, however, prevention by dibutyryl cAMP
was complete. This protective effect was significantly

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579
580
Figure 4 Post-translational alterations in hepatobiliary transporters in cholestasis
Cholestatic drugs may cis- or trans -inhibit BSEP/Bsep or induce endocytic retrieval of canalicular transporters. Cyclosporin A, glibenclamide, rifamycin, rifampicin,
bosentan, thiazolidinedione derivatives and flutamide all cis-inhibit either or both Bsep and BSEP, whereas PM4-S and E2 17G trans-inhibit the transporter in a
non-competitive manner. Alternatively, E2 17G inhibition can be explained by Mrp2 inhibitory modulation of Bsep transport activity via proteinprotein interaction, while
E2 17G is being transported by Mrp2. A number of cholestatic manoeuvres in rats also lead to endocytic internalization of canalicular transporters into the subapical
compartment (SAC); this may lead to delivery to the lysosomal compartment, followed by degradation. Canalicular transporter retrieval has also been confirmed in
several human cholestatic diseases (see text for details). Several signalling pathways have been proposed to mediate this retrieval in experimental models of cholestasis.
Activation of PKC, when activated by ethacrynic acid or TLC, induces transporter internalization in rats. In addition, activation of PKC in t -BOOH-induced oxidative
stress in rats leads to transporter retrieval as well. Experimental therapeutic manoeuvres may prevent the retrieval of the transporters or accelerate exocytic re-insertion.
Increases in intracellular cAMP levels induced by administration of the permeant cAMP analogue dibutyryl cAMP or by the phosphodiesterase inhibitor silibinin prevents
internalization and accelerates re-insertion, via cytosolic Ca2+ elevation, in rat cholestatic hepatocytes. On the other hand, TUDC prevents transporter endocytosis,
probably via ERK- and p38 MAPK-dependent pathways.
blocked by the Ca2+ chelator BAPTA/AM [1,2-bis-(oaminophenoxy)ethane-N,N,N ,N -tetra-acetic acid tetrakis(acetoxymethyl ester)], but not by the PKA (protein
kinase A) inhibitor KT5720, suggesting the involvement
of Ca2+ or Ca2+ -dependent signalling pathways in the
protective effect of dibutyryl cAMP. A similar protective
effect in terms of the signalling modulators involved was
afforded by silibinin, the active component of the hepatoprotector silymarin. This was most probably due to the
ability of silibinin to inhibit cAMP phosphodiesterase,
thus increasing endogenous cAMP levels [216].
TUDC (tauroUDC)
TUDC, the naturally occuring taurine conjugate of
UDC, stimulates exocytosis in the cholestatic liver [217]
and counteracts endocytic internalization of both Bsep
[218] and Mrp2 [196] in TLC-induced cholestasis. The
Ca2+ -sensitive PKC isoform PKC has been proposed
to mediate the anticholestatic effects of TUDC against
TLC-induced cholestasis [196]. This is in apparent
contradiction with more recent findings that PKC
is cholestatic rather than hepatoprotective [209]. In
previous studies, pan-specific PKC inhibitors were used,
and the biological response evoked by the interplay
between different PKC isoforms may be different from
that evoked by just one of them. Furthermore, TUDC

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activates the members of the MAPK (mitogen-activated

protein kinase) family ERK (extracellular-signalregulated kinase) [219] and p38 MAPK [220], and the
cholestatic effect of PKC may be overridden by the
choleretic effects of these signal transduction pathways.
CONCLUDING REMARKS AND FUTURE

DIRECTIONS
The recent enormous progress in molecular biology has
prompted the identification and characterization of several novel ATP-dependent and ATP-independent hepatocellular transporters, including many critically involved
in bile flow generation. This new information has been
quickly transferred to the liver physiopathology area,
providing an extensive understanding of the alterations
accounting for cholestatic diseases at the molecular level.
In acquired hepatocellular cholestasis, these alterations
may involve early changes in the dynamic localization
of canalicular transporters, followed by long-term transcriptional or post-transcriptional factors affecting
transporter synthesis. As a consequence of these primary
alterations, an adaptive response occurs to minimize the
detrimental effects of biliary compounds retained due
to the secretory failure. This includes decreased uptake,
increased biotransformation to less toxic metabolites,

decreased expression of canalicular transporters and
enhanced expression of alternative basolateral pumps,
which export these metabolites into the blood for further
renal excretion. This adaptive response is promoted by
the co-ordinated expression of a complex network of
nuclear receptors and hepatocyte-enriched transcription
factors, whose activity is modulated by biliary metabolites retained by the cholestatic failure. These adaptive
changes have been largely characterized in animal models
of experimental cholestasis. The occurrence of a similar
protective response in human cholestatic hepatopathies is
becoming increasingly apparent, but species differences
in the mechanisms of action (e.g. post-transcriptional in
humans compared with transcriptional in rodents)
appears to exist and will require characterization in each
case.
This adaptive spontaneous mechanism is, however,
not always sufficient to prevent hepatocellular damage
and therapeutic intervention is often required. A number
of newly designed therapeutic strategies are currently
being explored to enhance adaptability by targeted
activation of nuclear receptors with high-affinity specific
ligands. Furthermore, the recognition of the involvement
of these transcription factors in the anticholestatic
effects of remedies classically used in the treatment of
cholestatic disease (e.g. UDC and rifampicin) has added
a rationale to their usage and extended their applications.
The development of new tailor made strategies for
each hepatopathy by targeting specific nuclear receptors
according to the particular deficiency is a key future challenge for both basic and clinical hepatologists. This goal
will require characterization of the impairments in transporter and/or nuclear receptor expression in each human
cholestatic disease. The finding of more selective potent
nuclear receptor ligands for an appropriate gene control
will be also an area of active research in a near future.
Finally, a number of compounds have been tested
in animal models of cholestasis to prevent/revert the
endocytic internalization of canalicular transporters
that occurs shortly after cholestasis. These experimental
therapeutic strategies are based either on the wellrecognized capability of the therapeutic agents to favour
exocytic carrier re-insertion (e.g. dibutyryl cAMP and
TUDC) or to counteract potentially cholestatic signalling
pathways (e.g. PKC or PI3K inhibitors). Taking into
account that post-transcriptional changes involving
transporter retrieval are increasingly recognized in
cholestatic disease in humans and that they may account
for post-transcriptional changes in this hepatopathy,
this strategy may effectively complement the effect of
inducers of carrier expression by co-operatively assuring
proper localization and constitutive expression. Whether
such a concerted beneficial action will occur effectively
in patients with cholestatic hepatopathies remains to be
ascertained, and this will represent a challenge for clinical
researchers in the way to explore better therapeutic

alternatives in cholestatic liver disease.
ACKNOWLEDGMENTS
This work was supported by CONICET (Consejo
Nacional de Investigaciones Cientficas y Tecnicas; PIP
6442), and ANPCyT (Agencia Nacional de Promocion
Cientfica y Tecnologica; PICT 05-26115), Argentina.
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Received 3 July 2007/30 October 2007; accepted 7 November 2007

Published on the Internet 2 April 2008, doi:10.1042/CS20070227

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Hepatocellular Transport in Acquired Cholestasis: New Insights Into Functional, Regulatory and Therapeutic Aspects

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Clinical Science (2008) 114, 567588 (Printed in Great Britain) doi:10.

Hepatocellular transport in acquired

Marcelo G. ROMA, Fernando A. CROCENZI and Enrique A. SANCHEZ

an impairment of normal bile flow secondary to structural

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

inhibition of relevant transporters or changes in their

PHYSIOLOGICAL MECHANISMS OF BILE

The Authors Journal compilation

2008 Biochemical Society

Bile salts are the predominant organic solutes in

Hepatocellular transport in acquired cholestasis

by the secretin-regulated cAMP-dependent output of an

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

Changes in transporter expression in

Adaptive changes in the expression of transporters and

The Authors Journal compilation

2008 Biochemical Society

regulation of phase I and phase II bile-acid-metabolizing

Hepatocellular transport in acquired cholestasis

The bile salt conjugates and the bilirubin glucuronides

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

and of the apical transporter Mrp2 [38,51]; Mrp2 mRNA

The Authors Journal compilation

2008 Biochemical Society

diseases in humans. NTCP and OATP1B1 protein levels

Hepatocellular transport in acquired cholestasis

Role of nuclear receptors and hepatocyte-enriched

A similar FXR-mediated repression of the SHP/

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

condition appears to be due to a cytokine [IL (interleukin)

The Authors Journal compilation

2008 Biochemical Society

both FXR [129] and PPAR [130] increase bile acid

Changes in transporter expression as a primary cause of

Hepatocellular transport in acquired cholestasis

The decrease in canalicular transporter expression

Pure (bland) cholestasis

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

experimental EE-induced cholestasis in rats resembles

Manipulation of the expression of hepatocellular transporters

The Authors Journal compilation

2008 Biochemical Society

bile excretory route by stimulating defective transporter

Hepatocellular transport in acquired cholestasis

Ntcp [154]. Although FXR-mediated stimulation of Bsep

The Authors Journal compilation

2008 Biochemical Society

M. G. Roma, F. A. Crocenzi and E. A. Sanchez Pozzi

relevant acquired cholestasis in humans, such as PBC

The Authors Journal compilation

2008 Biochemical Society

Transporter inhibition by cholestatic drugs