Characterization of inducible nature of MRP3 in rat liver
Kotaro
Ogawa1,
Hiroshi
Suzuki1,5,
Tomoko
Hirohashi1,5,
Toshihisa
Ishikawa2,
Peter J.
Meier3,
Kenji
Hirose4,
Toshifumi
Akizawa4,
Masanori
Yoshioka4, and
Yuichi
Sugiyama1,5
1 Graduate School of Pharmaceutical Sciences,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;
2 Department of Experimental Pediatrics,
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030;
3 Division of Clinical Pharmacology and
Toxicology, Department of Medicine, University Hospital, CH-8091
Zurich, Switzerland; 4 Faculty of Pharmaceutical
Sciences, Setsunan University, Nagao-toge-machi, Hirakata, 573-01, Japan; and 5 Core Research for Evolutional
Sciences and Technology, Japan Science and Technology, Tokyo
113-0033, Japan
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ABSTRACT |
We found previously that expression of multidrug
resistance-associated protein (MRP) 3 is induced in a mutant rat strain
(Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and
in normal Sprague-Dawley (SD) rats after ligation of the common bile
duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na+-taurocholic
acid cotransporting polypeptide (Ntcp), organic anion transporting
polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and
primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and
MRP6] transporters.
-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas
expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment.
Although expression of MRP3 was also induced by administration of
phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by
any of these treatments. Moreover, the mRNA level of MRP3, but not that
of P-gp, was increased in SD rats after administration of bilirubin and
in Gunn rats whose hepatic bilirubin concentration is elevated because
of a defect in the expression of UDP-glucuronosyl transferase. However,
the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant
rats and in SD rats that had undergone common bile duct ligation or
-naphthylisothiocyanate treatment, we must assume that factor(s)
other than these physiological substances are also involved in the
increased protein level of MRP3.
induction; multidrug resistance-associated protein; canalicular
multispecific organic anion transporter; cholestasis; bilirubin
glucuronide
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INTRODUCTION |
THE LIVER IS ONE of the most important organs for the
detoxification of xenobiotics. Many compounds are metabolized in
hepatocytes after being taken up across the sinusoidal membrane and
then excreted into the bile across the bile canalicular membrane. Thus
transporters and metabolic enzymes have a synergistic action in the
detoxification of xenobiotics. Recently, cDNA cloning of transporters
for sinusoidal uptake and for canalicular export has been performed
(29, 31). The former include several kinds of secondary active
transporters such as Na+-taurocholic acid transporting
polypeptide (Ntcp) (1) and organic anion transporting polypeptide
(oatp) 1, which have been cloned as the transporters for the
Na+-dependent uptake of taurocholic acid and
Na+-independent uptake of organic anions, respectively (29,
31). As a homologue of oatp1, oatp2 has also been cloned and, indeed, it is capable of transporting organic anions (29, 31). Although OCT1,
an organic cation transporter, was initially cloned from kidney (23),
this transporter is also expressed on the sinusoidal membrane of
hepatocytes (30). The function of these transporters has been studied
by examining ligand uptake into cRNA-injected Xenopus oocytes
and/or into cDNA-transfected mammalian cells (29).
In the same manner, the molecular mechanisms for ligand export across
the canalicular membrane have also been identified. Several kinds of
primary active transporters have been shown to act as export pumps for
their ligands into bile; these belong to the ATP-binding cassette
transmembrane transporters (ABC transporters) and include the multidrug
resistance transporters mdr1 and -2, which are responsible for the
biliary excretion of cationic and neutral amphipathic compounds and
phospholipids, respectively (33, 36). In addition, certain bile acids
are also excreted into the bile via the canalicular bile salt export
pump, whose function is associated with the sister of P-glycoprotein
(P-gp) (10). Moreover, canalicular multispecific organic anion
transporter (cMOAT) has also been identified as a transporter involved
in the biliary excretion of many organic anions including conjugated xenobiotics. The substrate for cMOAT has been determined by comparing transport across the bile canalicular membrane between normal rats and
mutant rats whose cMOAT function is hereditarily defective; these
mutant rats include Groningen yellow, transport-deficient, and Eisai
hyperbilirubinemic rats (EHBR) (32, 38, 42). It has been shown that the
substrate specificity of cMOAT resembles that of multidrug
resistance-associated protein (MRP) 1; cMOAT is referred to as MRP2
(18, 31, 38). Although cMOAT/MRP2 is expressed on the bile canalicular
membrane (18), the expression of MRP1 in the liver is minimal.
Previously, we amplified the partial cDNA sequences of novel ABC
transporters that were initially referred to as MRP-like protein
(MLP)-1 and -2 (11). Subsequent sequence alignment revealed that MLP-1
and -2 are homologues of human MRP6 and MRP3, respectively (11, 25).
MRP6 is expressed in the liver of both SD rats and EHBR, whereas MRP3
is expressed only in EHBR liver (11). Moreover, hepatic expression of
MRP3 in normal rats was induced by common bile duct ligation (11). In
humans, it was also demonstrated that the hepatic expression of MRP3 is
induced under cholestatic conditions (24). Recent immunohistochemical
studies indicated that MRP3 is located on the basolateral membrane of
hepatocytes and cholangiocytes (24, 26). Because MRP3 can transport
organic anions including glucuronide conjugates (12), it is possible that MRP3 is induced to compensate for the impaired function of cMOAT/MRP2.
The present study has been undertaken to characterize the induction of
MRP3 under in vivo experimental conditions in relation to that of other
transporters responsible for hepatic uptake and biliary excretion.
Several kinds of inducers were used in the present study. Effect of
phenobarbital (PB) was examined, because we found that human MRP3 is
induced by PB in Hep G2 cells in vitro (19), and in addition, PB has
been reported to increase the biliary excretion of anionic compounds
and to induce the uptake of sulfobromophthalein into rat hepatocytes
(34). As an inducer, 3-methyl-cholanthrene (3-MC) was also used,
because the induction of P-gp by this reagent has been reported in rat
hepatocytes (6). Induction by 1-chloro-2,4-dinitrobenzene (CDNB) and
zinc was examined, because the former is conjugated intracellularly
with glutathione to yield dinitrophenyl-glutathione S
conjugate, a typical substrate for cMOAT/MRP2 (22) and the latter
induces MRP1 in the cisplatin-resistant human HL-60/R-CP cell line
(13). The expression of transporters under cholestatic conditions
induced by the administration of
-naphthylisothiocyanate (ANIT) was
also studied (20).
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METHODS |
Animals.
Male SD (220-260 g) and Wistar (220-240 g) rats were
purchased from Nihon Ikagaku Doubutsu (Shizuoka, Japan). Male EHBR
(200-250 g) were supplied by Eisai Laboratories (Gifu, Japan).
Male Gunn rats (180-220 g), established from Wistar rats as
mutants lacking UDP-glucuronosyl transferase activity, were purchased
from Sankyo Lab Service (Tokyo, Japan).
Drug treatment.
Rats received PB (80 mg/kg in saline ip, daily for 4 days), 3-MC (40 mg/kg in corn oil ip, daily for 3 days), ANIT (250 mg/kg in
polyethylene glycol, once po), CDNB (30 mg/kg in corn oil ip, daily for
3 days) or zinc acetate (6 mg/kg in saline iv, daily for 3 days).
Control rats received the respective vehicle. Livers were dissected 24 h after the final administration. Bilirubin was intravenously injected
into one group 3 times a day every 4 h for 2 days at a dose of 30 mg/kg, and livers were dissected 2 h after the final injection. All
livers were immediately frozen in liquid nitrogen and then transferred
to a freezer (
80°C) before preparation of mRNAs and membrane
fractions. The serum concentrations of bilirubin and
bilirubin-glucuronide were determined separately using a test kit (Wako
Pure Chemical, Tokyo, Japan) according to the alkaline azobilirubin method.
Northern blot analysis.
Specific probes for Ntcp, oatp1, OCT1, cMOAT/MRP2, and MRP1 for
Northern blot analysis were prepared from the sequence between bases
256 and 648 (393 bp) of rat Ntcp cDNA, the full length (2,200 bp) of
rat oatp1 cDNA, bases 549-1,090 (542 bp) of rat OCT1 cDNA, bases
3,972-4,392 (421 bp) of rat cMOAT/MRP2 cDNA, and a 421-bp fragment
(carboxy terminal ABC region) of rat MRP1 cDNA, respectively. The mdr
probe prepared from the sequence between bases 3,187 and 3,599 (413 bp)
of rat mdr1b cDNA could not distinguish mdr isoforms (mdr1a, -1b, and
-2). The MRP3 and -6 probes were prepared from the sequence between
bases 3,937 and 4,359 (423 bp) and between bases 3,875 and 4,296 (422 bp), respectively (11). Northern blot analysis was performed as
described previously (14). Two or five micrograms of
poly(A)+ RNA prepared from rat liver was separated on 0.8%
agarose gel containing 3.7% formaldehyde and transferred to a nylon
membrane (Biodyne; Pall, Glen Cove, NY) before fixation by baking for 2 h at 80°C. Blots were prehybridized in hybridization buffer
containing 4× SSC (1× SSC consists of 0.15 M NaCl and 0.15 M sodium citrate, pH 7.0), 5× Denhardt's solution, 0.2% SDS,
0.1 mg/ml sonicated salmon sperm DNA, and 50% formamide at 42°C
for 2 h. Hybridization was performed overnight in the same buffer
containing 106 cpm/ml 32P-labeled cDNAs
prepared by a random primed labeling method. As a control to ensure
equal gel loading of mRNAs, 32P-labeled cDNA of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech Laboratories,
Palo Alto, CA) was used. The hybridized membrane was washed in 2×
SSC-0.1% SDS at room temperature for 20 min, followed by washing in
2× SSC-0.1% SDS at 55°C for 20 min and then in 0.1×
SSC-0.1% SDS at 55°C for 20 min. The membranes were exposed to an
imaging plate (Fuji Photo Film, Tokyo, Japan) for a period ranging from
3 h to overnight. The relative induction ratio was defined as the
intensity of specific bands of the treated group to that of the control
group after normalization for the intensity of GAPDH mRNA. The
detection limit of band intensity was assumed to be 20% of the
background radioactivity detected on an imaging plate.
Antibodies for Western blot analysis.
Polyclonal anti-rat Ntcp antibody and EAG15 polyclonal anti-rat
cMOAT/MRP2 antibody described previously were used in the present
study. C219 monoclonal anti-mouse P-gp antibody was purchased from
Centcor (Malvern, PA). The polyclonal anti-rat MRP3 antibody was raised
against a maltose-binding protein fusion protein containing the 136 amino acids corresponding to bases 838-973 of the deduced rat MRP3
amino acid sequence. The pMAL-c2 expression vector (New England
Biolabs, Beverly, MA) was used for the expression of the fusion
protein. After purification by amylose resin, rabbits were immunized
with 250 µg of the fusion protein mixed with Freund's complete
adjuvant (Sigma).
Membrane preparation.
Crude liver membranes were prepared by the method of Gant et al. (6).
Liver was homogenized in 5 vols of 0.1 M Tris · HCl buffer (pH 7.4) containing 1 µg/ml leupeptin and pepstatin A and 50 µg/ml phenylmethylsulfonyl fluoride with 20 strokes of a Dounce homogenizer. After centrifugation (1,500 g for 10 min), the
homogenate supernatant was centrifuged at 100,000 g for 30 min.
The precipitate was suspended in Tris · HCl buffer
and again centrifuged at 100,000 g for 30 min. The crude
membrane fraction was resuspended in 0.1 M Tris · HCl
buffer (pH 7.4) containing the proteinase inhibitors using five strokes
of a Dounce homogenizer. Plasma membranes were prepared as described
previously (22). Briefly, livers were homogenized in buffer A
containing 250 mM sucrose, 1 mM EGTA, and 5 mM HEPES (pH 7.4) with a
Dounce homogenizer. After centrifugation of the homogenate at 1,500 g for 15 min, the resulting pellet was suspended in buffer
A and Percoll (Pharmacia Biotech, Uppsala, Sweden), and centrifuged
at 30,000 g for 60 min. The turbid layer was suspended in
buffer B containing 250 mM sucrose and 50 mM Tris · HCl (pH 7.4) and centrifuged at 8,000 g for 10 min. The resulting pellet was suspended in buffer
B, homogenized with a Dounce homogenizer, and layered over 38%
sucrose. After centrifugation at 16,000 g for 70 min, the
interfaces were collected, washed by centrifugation at 75,000 g
for 30 min in buffer B, and suspended in buffer B using
a Teflon homogenizer. All procedures were performed at 0-4°C.
The crude and plasma membranes were stored at
80°C before
being used for Western blot analysis. The membrane protein concentrations were assayed according to the method of Bradford (3).
Western blot analysis.
Crude membrane (25 µg) or plasma membrane (20 µg) was dissolved in
10 µl of 0.25 M Tris · HCl buffer containing 2%
SDS, 30% glycerol, and 0.01% bromophenol blue (pH 6.8), without
boiling, and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel with a 4.4% stacking gel. Proteins were transferred
electrophoretically to nitrocellulose membranes (Immobilon; Millipore,
Bedford, MA) using a blotter (Trans-blot; Bio-Rad, Richmond, CA) at 15 V for 1 h. The membranes were blocked with Tris-buffered saline
containing 0.05% Tween 20 (TBS-T) and 5% BSA for 1-2 h at room
temperature. After being washed with TBS-T (3 × 5 min), the
membranes were incubated with the following concentrations of primary
antibodies in TBS-T containing 5% BSA overnight at 4°C and then
washed with TBS-T (3 × 5 min): polyclonal anti-rat Ntcp serum
(dilution 1:5,000) (16), EAG15 (1:10,000) (4), C219 (1 µg/ml), or
polyclonal anti-rat MRP3 antibody (1:200). The membranes were allowed
to bind 125I-labeled sheep anti-rabbit IgG antibody for
Ntcp and cMOAT/MRP2 or sheep anti-mouse IgG antibody for P-gp diluted
1:200 in TBS-T containing 5% BSA for 1 h at room temperature and then
were placed in contact with an imaging plate for a period ranging from
3 h to overnight after being washed with TBS-T (3 × 5 min). The
intensity of specific bands was quantified from a standard curve using
a BAS 2000 system (Fuji Photo Film, Tokyo, Japan). The relative induction ratio was defined as the ratio of the intensity of a specific
band of the treated group to that of the control group.
Statistical analysis.
Statistical analysis of differences between control and treated groups
was performed by paired t-test. Statistical significance was
taken as a P value of <0.05. Unless otherwise stated, all data represent means ± SE of three to five animals.
 |
RESULTS |
Expression of transporters in drug-treated SD rats.
The expression levels of transcripts of transporters after different
treatments were examined by Northern blot analysis (Fig. 1 and Table
1). Oatp1 mRNA was significantly reduced
by ANIT treatment and bile duct ligation to 47% (P < 0.01)
and 33% (P < 0.01) of the control, respectively (Fig. 1 and
Table 1). Ntcp mRNA was also reduced by treatment with PB, ANIT, and
bile duct ligation to 84% (P < 0.05), 69% (P < 0.05) and 36% (P < 0.01) of the control, respectively (Fig.
1 and Table 1). OCT1 mRNA was reduced to 64% (P < 0.01) only by bile duct ligation (Fig. 1 and Table 1).


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Fig. 1.
Typical patterns of Northern blot analysis of mRNAs of transporters in
male Sprague-Dawley (SD) rat liver after drug treatment. Hepatic
expression of transporters was examined in male SD rats that received
phenobarbital (PB; 80 mg/kg ip for 4 days), 3-methylcholanthrene (3-MC;
40 mg/kg ip for 3 days), -naphthylisothiocyanate (ANIT; 250 mg/kg
po, single dose), 1-chloro-2,4-dinitrobenzene (CDNB; 30 mg/kg ip for 3 days), zinc acetate (6 mg/kg iv for 3 days) and bile duct ligation
(BDL; for 3 days). Two micrograms of mRNA were applied to each lane.
Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used
as a control for equal loading. Ntcp, Na+-taurocholic acid
contransporting polypeptide; oatp1, organic anion transporting
polypeptide; OCT1, organic cation transporter; mdr, multidrug
resistance transporter; cMOAT, canalicular multispecific organic anion
transporter; MRP, multidrug resistance-associated protein.
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No significant change was observed in the level of cMOAT/MRP2 mRNA
after any of the treatments, except for a slight reduction to 80%
(P > 0.05) of the control after bile duct ligation (Fig. 1
and Table 1). P-gp mRNA was induced 4.8-fold (P < 0.01) and 8.2-fold (P < 0.01) by ANIT treatment and bile duct ligation, respectively (Fig. 1 and Table 1). MRP6 mRNA was significantly reduced
by treatment with PB and ANIT and by bile duct ligation to 51% (P
< 0.05), 69% (P < 0.05), and 65% (P < 0.05)
of the control, respectively (Fig. 1 and Table 1). In contrast, MRP3
mRNA was induced >4.6-fold (P < 0.01), 5.9-fold (P
< 0.01), and 6.1-fold (P < 0.01) after PB and ANIT
treatment and bile duct ligation, respectively, whereas MRP3 mRNA was
at marginal levels in untreated SD rats (Figs. 1 and
2 and Table 1).

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Fig. 2.
Typical patterns of Western blot analysis of transporters in male SD
rat liver after drug treatment. Hepatic expression of transporters was
examined in male SD rats that received PB (80 mg/kg ip for 4 days),
ANIT (250 mg/kg po, single dose) and BDL (for 3 days). Twenty-five
micrograms of protein of crude membrane fraction prepared from liver
were applied to each lane; P-gp, P-glycoprotein.
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The expression level of transporter proteins was determined by Western
blot using membrane fractions and specific antibodies (Fig. 2 and Table
1). Both Ntcp protein (43 kDa) and cMOAT/MRP2 protein (190 kDa) were
significantly reduced to 60% (P < 0.05) and 59%
(P < 0.05) of the control after bile duct ligation,
respectively (Fig. 2 and Table 1). In contrast, P-gp protein (170 kDa)
was markedly induced by 10-fold (P < 0.01) and
22-fold (P < 0.01) after ANIT treatment and bile duct
ligation, respectively (Fig. 2 and Table 1). PB treatment did not
affect the expression of Ntcp, cMOAT/MRP2, and P-gp at all protein
levels (Fig. 2 and Table 1). MRP3 protein (~170 kDa) was markedly
induced by 16-fold (P < 0.01), 4.8-fold (P < 0.05),
and 3.8-fold (P < 0.05) by bile duct ligation and PB and ANIT
treatment, respectively (Fig. 2 and Table 1). The increase in
transporter proteins by these treatments was well correlated with the
increased mRNA levels (Figs. 1 and 2 and Table 1).
Relationship between serum levels of bilirubin and/or its
glucuronides and MRP3 induction.
Because the expression of MRP3, but not that of P-gp, was induced in
EHBR, whose bilirubin and glucuronide levels are elevated in serum
(Fig. 3 and Table
2) (11), we examined the hypothesis that
these endogenous compounds could be associated with the induction of
MRP3. Indeed, the increased expression of MRP3 by ANIT treatment and
bile duct ligation (Figs. 1 and 2) was associated with the elevated
plasma levels of bilirubin and its glucuronide.

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Fig. 3.
Typical patterns of Northern blot analysis of mRNAs of P-gp and MRP3 in
Eisai hyperbilirubinemic rat (EHBR) liver. Five micrograms of mRNA from
normal rat (SD rat) liver (control), EHBR liver (EHBR), and liver from
rats after BDL were hybridized with cDNA probes for rat P-gp, MRP3, and
GAPDH as a control to ensure equal loading.
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Table 2.
Relationship between serum bilirubin and bilirubin-glucuronide
concentrations and expression of mRNAs and proteins of transporters in
liver of EHBR, Gunn, and drug-treated SD rats
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The expression of MRP3 mRNA in the liver of Gunn rats was also measured
to examine whether unconjugated bilirubin was involved in the induction
of this transporter. In Gunn rats, the serum concentration of
bilirubin, rather than its glucuronide, was markedly higher (5.5 mg/dl)
than in normal rats (Table 2). MRP3 mRNA was found to be significantly
increased to 3.4-fold (P < 0.05) of the control level (Fig.
4 and Table 2). The expression of P-gp mRNA
in Gunn rats was comparable to that in normal rats (Fig. 4 and Table
2).

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Fig. 4.
Typical patterns of Northern blot analysis of mRNA of P-gp and MRP3 in
Gunn rat liver. Five micrograms of mRNA from normal rat (Wistar rat)
liver (control), Gunn rat liver, and liver of rats subjected to BDL
were hybridized with cDNA probes for rat P-gp, MRP3, and GAPDH as a
control to ensure equal loading.
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The expression of MRP3 was then examined in SD rats after an
intravenous injection of bilirubin. The serum bilirubin concentration rose markedly (~8 mg/dl) immediately after treatment and
then declined rapidly to a normal level (~0.3 mg/dl) within 2 h after each treatment (Table 2). Serum bilirubin glucuronide was also elevated, ranging from 0.4 to 2 mg/dl. Hepatic MRP3 mRNA was induced 3.1-fold (P < 0.05) in liver from bilirubin-treated SD rats
(Fig. 5 and Table 2). On the other hand,
P-gp mRNA did not change significantly after bilirubin treatment,
although marked induction was observed after bile duct ligation (Fig. 5
and Table 2). The protein level of MRP3, however, was not affected by
bilirubin administration (Fig. 6).

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Fig. 5.
Typical patterns of Northern blot analysis of mRNAs of P-gp and MRP3 in
bilirubin-treated rat liver. Bilirubin (30 mg/kg iv) was administered 3 times/day for 2 days to SD rats. Five micrograms of mRNA from SD rat
liver (control), liver of bilirubin-treated animals, and liver of
animals subjected to BDL were hybridized with cDNA probes for rat P-gp,
MRP3, and GAPDH as a control to ensure equal loading.
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Fig. 6.
Typical patterns of Western blot analysis of MRP3 in bilirubin-treated
rat liver. Bilirubin (30 mg/kg iv) was administered 3 times/day for 2 days to SD rats. Twenty-five micrograms of protein of crude membrane
fraction prepared from liver were applied to each lane.
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DISCUSSION |
In the present study, we determined the expression levels of hepatic
transporters that are responsible for the uptake and export into the
bile. In Northern blot analysis, the mRNA level of transporters was
corrected by that of GAPDH. Because the expression level of GAPDH may
also be affected by several kinds of treatments, the results may
contain some bias. In the cholestatic rat liver, downregulation of Ntcp
(7, 8) oatp1 (9), and cMOAT/MRP2 (39), along with induction of P-gp
(35), have been reported. In the present study, the expression of Ntcp
and oatp1 was significantly reduced by ANIT and bile duct ligation
(Fig. 1 and Table 1). The reduced expression of Ntcp and oatp1 in the
cholestatic liver is consistent with the previous observations
(7-9, 40). The molecular mechanism reported by Trauner et al. (40)
may be related to the downregulation of Ntcp. At the present time, the
mechanism for the reduced expression of oatp1 still remains to be
clarified; although the sequence of the promoter region of human OATP
was reported, the element controlling the transcription of oatp1 is different from that for OATP (27).
In contrast to these transporters, the expression of P-gp and MRP3 was
markedly induced by ANIT treatment and by bile duct ligation (Fig. 1
and Table 1). The upregulation of P-gp under cholestatic conditions is
consistent with previous findings (35). Moreover, MRP3 was also
markedly induced by PB treatment (Fig. 1 and Table 1). These
observations contrast with the fact that neither of these treatments
affected the mRNA levels of cMOAT/MRP2 and MRP6 (Fig. 1 and Table 1).
The reduced cMOAT/MRP2 protein after the bile duct ligation is
consistent with previous observations (39, 41). Although rat and human
cMOAT/MRP2 is induced by PB in cultured cells in vitro (17, 19), PB was
not effective in in vivo studies (Fig. 1 and Table 1). The same
discrepancy was observed for 3-MC; 3-MC induced rat mdr1 in vitro
(6) but not in vivo (Fig. 1 and Table 1). The difference between in
vitro and in vivo inducibility may be ascribed to the difference in drug exposure, although the administered doses were sufficient to
induce the hepatic expression of metabolic enzymes (2).
We could not distinguish the expression levels of mdr isoforms by using
the present cDNA fragment and antibody. Using a much more sophisticated
method, Schrenk et al. (35) indicated that both mdr1a and -1b,
but not mdr2, were markedly induced by ANIT treatment and by bile duct
ligation and that the expression level of mdr1b in the treated liver
was much higher than that of mdr1a (35). Moreover, Vos et al. (41)
reported the downregulated expression of sister of P-gp, the increased
expression of mdr1b, and unchanged expression of mdr1a and mdr2 in
endotoxin-treated rats. These results are consistent with the
hypothesis that the marked induction of P-gp observed in the present
study (Fig. 1 and Table 1) may represent the induction of mdr1b.
The present study clearly demonstrates that hepatic MRP3 expression is
induced in normal rat liver after treatment with PB, ANIT, or bile duct
ligation (Figs. 1 and 2), as with untreated EHBR. Because
hyperbilirubinemia was associated with ANIT treatment and bile duct
ligation, as with untreated EHBR (Table 2), it was hypothesized that
bilirubin and/or its glucuronide may participate in the induction of
MRP3. Although PB induced MRP3, the plasma concentration of bilirubin
and its glucuronide remained normal after administration of PB (data
not shown), suggesting that PB activates the transcription of MRP3 by
another mechanism(s). It is possible that some nuclear receptors (such
as the pregnane X receptor) may be involved in this induction, as
reported for the cytochrome P-450 3A enzymes (21, 28). To
examine the role of bilirubin and/or bilirubin glucuronides in the
induction, we determined the expression of MRP3 in Gunn rats with
higher serum bilirubin concentrations caused by a hereditary defect in
the expression of enzymes responsible for the glucuronide conjugation of bilirubin (UDP-glucuronosyltransferase) (15). The expression of MRP3
was enhanced in Gunn rats, in which markedly increased bilirubin, with
an increase in bilirubin glucuronide to a lesser extent, was observed
(Fig. 4 and Table 2). In the same manner, after administration of
bilirubin, marked increase in bilirubin, with a marginal increase in
its glucuronides, was also observed (Table 2), which was associated
with the increase in the mRNA level of MRP3. These results are
consistent with the hypothesis that bilirubin and/or its glucuronide
may be related to the transcriptional control of MRP3. The previous
finding that bilirubin is an endogenous substrate for aryl hydrocarbon
receptor (37) is consistent with the hypothesis that bilirubin and/or
bilirubin glucuronide may be related to the regulation of
transcription. Although MRP3 is also induced in human liver under
cholestatic conditions (24), the mechanism may be different from that
for rat MRP3, because the xenobiotic responsible element is not located
in the 5'-flanking region of human MRP3 (5). The protein level of
MRP3, however, was not significantly affected by the administration of
bilirubin (Fig. 6). To account for these data, we have to assume that
factor(s) other than bilirubin and/or its glucuronide are also involved in the increased protein level of MRP3. The precise mechanism for the
posttranscriptional regulation of MRP3 still remains to be clarified.
In contrast to MRP3, P-gp was not induced in either Gunn rats or EHBR
(Figs. 4 and 5 and Table 2), irrespective of the fact that the highest
induction of P-gp was observed after ANIT treatment and bile duct
ligation. The mechanism for P-gp induction under cholestatic conditions
may be different from that of MRP3, because P-gp was not induced in
either Gunn rats or EHBR (Figs. 4 and 5 and Table 2). These results
agree with the report that mdr1a and -1b are not induced by bilirubin
in primary cultured rat hepatocytes (35).
In conclusion, the results of the present study suggest that MRP3
expression is induced under cholestatic conditions. Together with the
recent finding that MRP3 transports organic anions including glucuronide conjugates, it is possible that MRP3 can compensate for the
impaired expression of cMOAT/MRP2 in EHBR. Although it was found that
the increased mRNA level of MRP3 was associated with the increased
concentration of bilirubin and/or its glucuronides in mutant rats and
in SD rats that had undergone common bile duct ligation or ANIT
treatment, administration of bilirubin resulted in the increase in the
level of mRNA, but not the protein level, of MRP3. It was suggested
that we must assume that factor(s) other than these physiological
substances are also involved in the increased protein level of MRP3.
Identification of this factor(s) still remains to be clarified.
 |
ACKNOWLEDGEMENTS |
We express our appreciation to Dr. D. Keppler from the Deutsches
Krebsforschungszentrum (Heidelberg, Germany) for the kind gift of
polyclonal antibody EAG15, which detects cMOAT/MRP2.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Sugiyama,
Graduate School of Pharmaceutical Sciences, Univ. of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail:
sugiyama{at}seizai.f.u-tokyo.ac.jp).
Received 19 March 1999; accepted in final form 26 October 1999.
 |
REFERENCES |
1.
Ananthanarayanan, M.,
O. C. Ng,
J. L. Boyer,
and
F. J. Suchy.
Characterization of cloned rat liver Na+-bile acid cotransporter using peptide and fusion protein antibodies.
Am. J. Physiol. Gastrointest. Liver Physiol.
267:
G637-G643,
1994[Abstract/Free Full Text].
2.
Bock, K. W.,
H. P. Lipp,
and
B. S. Bock-Hennig.
Induction of drug-metabolizing enzymes by xenobiotics.
Xenobiotica
20:
1101-1111,
1990[ISI][Medline].
3.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[ISI][Medline].
4.
Buchler, M.,
J. Konig,
M. Brom,
J. Kartenbeck,
H. Spring,
T. Horie,
and
D. Keppler.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats.
J. Biol. Chem.
271:
15091-15098,
1996[Abstract/Free Full Text].
5.
Fromm, M. F.,
B. Leake,
D. M. Roden,
G. R. Wilkinson,
and
R. B. Kim.
Human MRP3 transporter: identification of the 5'-flanking region, genomic organization and alternative splice variants.
Biochim. Biophys. Acta
1415:
369-374,
1999[ISI][Medline].
6.
Gant, T. W.,
J. A. Silverman,
H. C. Bisgaard,
R. K. Burt,
P. A. Marino,
and
S. S. Thorgeirsson.
Regulation of 2-acetylaminofluorene- and 3-methylcholanthrene-mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver.
Mol. Carcinog.
4:
499-509,
1991[ISI][Medline].
7.
Gartung, C.,
M. Ananthanarayanan,
M. A. Rahman,
S. Schuele,
S. Nundy,
C. J. Soroka,
A. Stolz,
F. J. Suchy,
and
J. L. Boyer.
Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis.
Gastroenterology
110:
199-209,
1996[ISI][Medline].
8.
Gartung, C.,
S. Schuele,
S. F. Schlosser,
and
J. L. Boyer.
Expression of the rat liver Na+/taurocholate cotransporter is regulated in vivo by retention of biliary constituents but not their depletion.
Hepatology
25:
284-290,
1997[ISI][Medline].
9.
Gartung, C.,
M. Trauner,
S. F. Schlosser,
B. Hagenbuch,
P. J. Meier,
and
J. L. Boyer.
Sodium-independent uptake of bile acids is unaffected by down-regulation of an organic anion transporter (oatp) in rat liver during cholestasis produced by common bile duct ligation (CBDL) (Abstract).
Hepatology
24:
369A,
1997.
10.
Gerloff, T.,
B. Stieger,
B. Hagenbuch,
J. Madon,
L. Landmann,
J. Roth,
A. F. Hofmann,
and
P. J. Meier.
The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver.
J. Biol. Chem.
273:
10046-10050,
1998[Abstract/Free Full Text].
11.
Hirohashi, T.,
K. Ito,
K. Ogawa,
H. Suzuki,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Hepatic expression of multidrug resistance-associated protein (MRP)-like proteins maintained in Eisai hyperbilirubinemic rats (EHBR) Mol.
Pharmacol.
53:
1068-1075,
1998.
12.
Hirohashi, T.,
K. Suzuki,
and
Y. Sugiyama.
Characterization of the transport properties of cloned rat multidrug resistance associated protein 3 (MRP3).
J. Biol. Chem.
274:
15181-15485,
1999[Abstract/Free Full Text].
13.
Ishikawa, T.,
J. J. Bao,
Y. Yamane,
K. Akimaru,
K. Frindrich,
C. D. Wright,
and
M. T. Kuo.
Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells.
J. Biol. Chem.
271:
14981-14988,
1996[Abstract/Free Full Text].
14.
Ito, K.,
H. Suzuki,
T. Hirohashi,
K. Kume,
T. Shimizu,
and
Y. Sugiyama.
Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR.
Am. J. Physiol. Gastrointest. Liver Physiol.
272:
G16-G22,
1997[Abstract/Free Full Text].
15.
Iyanagi, T.,
Y. Emi,
and
S. Ikushiro.
Biochemical and molecular aspects of genetic disorders of bilirubin metabolism.
Biochim. Biophys. Acta
1407:
173-184,
1998[ISI][Medline].
16.
Jacquemin, E.,
B. Hagenbuch,
B. Stieger,
A. W. Wolkoff,
and
P. J. Meier.
Expression cloning of a rat liver Na(+)-independent organic anion transporter.
Proc. Natl. Acad. Sci. USA
91:
133-137,
1994[Abstract].
17.
Kauffmann, H. M.,
and
D. Schrenk.
Sequence analysis and functional characterization of the 5'-flanking region of the rat multidrug resistance protein 2 (mrp2) gene.
Biochem. Biophys. Res. Commun.
245:
325-331,
1998[ISI][Medline].
18.
Keppler, D.,
and
J. Konig.
Hepatic canalicular membrane 5: expression and localization of the conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver.
FASEB J.
11:
509-516,
1997[Abstract/Free Full Text].
19.
Kiuchi, Y.,
H. Suzuki,
T. Hirohashi,
C. A. Tyson,
and
Y. Sugiyama.
cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3).
FEBS Lett.
433:
149-152,
1998[ISI][Medline].
20.
Klaassen, C. D.,
and
J. B. Watkins.
Mechanisms of bile formation, hepatic uptake, and biliary excretion.
Pharmacol. Rev.
36:
1-67,
1984[ISI][Medline].
21.
Kliewer, S. A.,
J. T. Moore,
L. Wade,
J. L. Staudinger,
M. A. Watson,
S. A. Jones,
D. D. McKee,
B. B. Oliver,
T. M. Willson,
R. H. Zetterstrom,
T. Perlmann,
and
J. M. Lehmann.
An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.
Cell
92:
73-82,
1998[ISI][Medline].
22.
Kobayashi, K.,
Y. Sogame,
H. Hara,
and
K. Hayashi.
Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membranes.
J. Biol. Chem.
265:
7737-7741,
1990[Abstract/Free Full Text].
23.
Koepsell, H.
Organic cation transporters in intestine, kidney, liver and brain.
Annu. Rev. Physiol.
60:
243-266,
1998[ISI][Medline].
24.
Konig, J.,
D. Rost,
Y. Cui,
and
D. Keppler.
Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane.
Hepatology
29:
1156-1163,
1999[ISI][Medline].
25.
Kool, M.,
M. de Haas,
G. L. Scheffer,
R. J. Scheper,
M. J. van Eijk,
J. A. Juijn,
F. Baas,
and
P. Borst.
Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.
Cancer Res.
57:
3537-3547,
1998[Abstract].
26.
Kool, M.,
M. van der Linden,
M. de Haas,
G. L. Scheffer,
J. M. de Vree,
A. J. Smith,
G. Jansen,
G. J. Peters,
N. Ponne,
R. J. Scheper,
R. P. Elferink,
F. Baas,
and
P. Borst.
MRP3, an organic anion transporter able to transport anti-cancer drugs.
Proc. Natl. Acad. Sci. USA
96:
6914-6919,
1999[Abstract/Free Full Text].
27.
Kullak-Ublick, G. A.,
U. Beuers,
C. Fahney,
B. Hagenbuch,
P. J. Meier,
and
G. Paumgartner.
Identification and functional characterization of the promoter region of the human organic anion transporting polypeptide gene.
Hepatology
26:
991-997,
1997[Medline].
28.
Lehmann, J. M.,
D. D. McKee,
M. A. Watson,
T. M. Willson,
J. T. Moore,
and
S. A. Kliewer.
The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.
J. Clin. Invest.
102:
1016-1023,
1998[Abstract/Free Full Text].
29.
Meier, P. J.
Hepatocellular transport systems: from carrier identification in membrane vesicles to cloned proteins.
J. Hepatol.
24, Suppl.1:
29-35,
1996[ISI][Medline].
30.
Meyer-Wentrup, F.,
U. Karbach,
V. Gorboulev,
P. Arndt,
and
H. Koepsell.
Membrane localization of the electrogenic cation transporter rOCT1 in rat liver.
Biochem. Biophys. Res. Commun.
248:
673-378,
1998[ISI][Medline].
31.
Müller, M.,
and
P. L. Jansen.
Molecular aspects of hepatobiliary transport.
Am. J. Physiol. Gastrointest. Liver Physiol.
272:
G1285-G1303,
1997[Abstract/Free Full Text].
32.
Oude Elferink, R. P.,
D. K. Meijer,
F. Kuipers,
P. L. Jansen,
A. K. Groen,
and
G. M. Groothuis.
Hepatobiliary secretion of organic compounds: molecular mechanisms of membrane transport.
Biochim. Biophys. Acta
1241:
215-268,
1995[ISI][Medline].
33.
Oude Elferink, R. P.,
G. N. Tytgat,
and
A. K. Groen.
Hepatic canalicular membrane 1: the role of mdr2 P-glycoprotein in hepatobiliary lipid transport.
FASEB J.
11:
19-28,
1997[Abstract/Free Full Text].
34.
Potter, B. J.,
J. Z. Ni,
K. Wolfe,
D. Stump,
and
P. D. Berk.
Induction of a dose-related increase in sulfobromophthalein uptake velocity in freshly isolated rat hepatocytes by phenobarbital.
Hepatology
20:
1078-1085,
1994[ISI][Medline].
35.
Schrenk, D.,
T. W. Gant,
J. A. Silverman,
K. H. Preisegger,
P. A. Marino,
and
S. S. Thorgeirsson.
Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates.
Hepatology
17:
854-860,
1993[ISI][Medline].
36.
Silverman, J. A.,
and
D. Schrenk.
Hepatic canalicular membrane 4: expression of the multidrug resistance genes in the liver.
FASEB J.
11:
308-313,
1997[Abstract/Free Full Text].
37.
Sinal, C. J.,
and
J. R. Bend.
Aryl hydrocarbon receptor-dependent induction of cyp1a1 by bilirubin in mouse hepatoma hepa1c1c7 cells.
Mol. Pharmacol.
52:
590-599,
1997[Abstract/Free Full Text].
38.
Suzuki, H.,
and
Y. Sugiyama.
Excretion of GSSG and gluthathione conjugates mediated by MRP1 and cMOAT/MRP2.
Semin. Liver Dis.
18:
359-376,
1998[ISI][Medline].
39.
Trauner, M.,
M. Arrese,
C. J. Soroka,
M. Ananthanarayanan,
T. A. Koeppel,
S. F. Schlosser,
F. J. Suchy,
D. Keppler,
and
J. L. Boyer.
The rat canalicular conjugate export pump (MRP2) is down-regulated in intrahepatic and obstructive cholestasis.
Gastroenterology
113:
255-264,
1997[ISI][Medline].
40.
Trauner, M.,
M. Arrese,
H. Lee,
J. L. Boyer,
and
S. J. Karpen.
Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors.
J. Clin. Invest.
101:
2092-2100,
1998[Abstract/Free Full Text].
41.
Vos, T. A.,
G. J. E. J. Hooiveld,
H. Koning,
S. Childs,
D. K. F. Meijer,
H. Moshage,
P. L. M. Jansen,
and
M. Müller.
Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, spgp, in endotoxemic rat liver.
Hepatology
28:
1637-1644,
1998[ISI][Medline].
42.
Yamazaki, M.,
H. Suzuki,
and
Y. Sugiyama.
Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics.
Pharm. Res.
13:
497-513,
1996[ISI][Medline].
Am J Physiol Gastroint Liver Physiol 278(3):G438-G446
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