Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells.

Abstract

CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na+-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via K(ATP) channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a K(ATP) channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)-CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane.

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BILE ACIDS ALTER THE SUBCELLULAR LOCALIZATION OF CNT2 AND INCREASE
CNT2-RELATED TRANSPORT ACTIVITY IN LIVER PARENCHYMAL CELLS.

Sonia Fernández-Veledo*, Isabel Huber-Ruano, Ivette Aymerich, Sylvie Duflot, F.Javier
Casado and Marçal Pastor-Anglada


Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Diagonal 645, E-
08028 Barcelona, Spain.
* Present address: Departamento de Bioquímica y Biología Molecular-II, Universidad
Complutense, E-28040 Madrid, Spain

Address for correspondence:
Marçal Pastor-Anglada
Departament de Bioquímica i Biologia Molecular
Facultat de Biologia. Universitat de Barcelona.
Diagonal 645, E-08028 Barcelona, Spain
Phone#34934021543
Fax#34934021559
E-mail address: mpastor@ub.edu

Running title: Bile acids increase CNT2-related transport activity
Keywords: liver parenchymal cells, plasma membrane transporters, CNT2, adenosine,
trafficking, bile acids
Number of figures: 7

Contract grant sponsor: Ministerio de Educación y Ciencia, Spain; Generalitat de Calunya
(Spain).
Contract grant number: SAF2002-0717
I.H.-R. and I.A hold a fellowship from Ministerio de Educación y Ciencia (Spain). S.F.-V. held a
fellowship from Ministerio de Ciencia y Tecnología (Spain) and S.D. was supported by the
University of Barcelona.
Biochemical Journal Immediate Publication. Published on 3 Jan 2006 as manuscript BJ20051232
Copyright 2006 Biochemical Society

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SUMMARY

CNT2 is a high-affinity Na+-coupled adenosine plasma membrane transporter, also
localized in intracellular structures. This transporter protein may play additional roles other than
nucleoside salvage, since it has recently been shown to be under purinergic control, via KATP
channels, by a mechanism that does not seem to involve changes in its subcellular localization.
In an attempt to identify agents that promote CNT2 trafficking, bile acids were found to increase
CNT2-related transport activity, in both Fao hepatoma and rat liver parenchymal cells, in a KATP
channel-independent manner. Maximum effect was recorded after treatment with hydrophilic
acids such as taurocholate (TCA). This effect did not involve changes in CNT2 protein amounts,
rather it was associated with a subcellular redistribution of CNT2, resulting in accumulation of
the transporter at the plasma membrane. This was deduced from subcellular fractionation studies,
biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin
precipitates and in vivo confocal microscopy analysis of a YFP-CNT2 construct. The induction
of CNT2, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059, and
colchicine, thus suggesting the involvement of the PI3-kinase/ERK pathway in a microtubule-
dependent activation of rCNT2. These effects are novel to bile-acid physiology and provide the
first evidence of short-term regulated translocation of CNT2 into/from the plasma membrane.






Biochemical Journal Immediate Publication. Published on 3 Jan 2006 as manuscript BJ20051232
Copyright 2006 Biochemical Society

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INTRODUCTION

CNT2 (SLC28A2) is a Na+-coupled concentrative transporter that preferentially
translocates purine nucleosides across the plasma membrane (for recent updated reviews see [1-
3]). CNT2 cDNA was initially isolated by expression cloning in Xenopus oocytes using a rat
liver cDNA library and was originally called SPNT, from Sodium-Purine Nucleoside Transporter
[4]. However, CNT2 is expressed in most epithelia and in cells of the immune system [5-7]. In
liver parenchymal cells, it is located at the sinusoidal pole, and in intracellular compartments
[8]. CNT2 mRNA is up-regulated during cell-cycle progression in synchronized cultured rat
hepatoma Fao cells induced to proliferate [9], and CNT2 protein amounts are increased in
regenerating rat livers soon after hepatectomy [10]. Moreover, CNT2 is expressed in
differentiated hepatocytes and, accordingly, its expression is lost in chemically-induced
hepatocarcinogenesis [11].
Recent data on CNT2 regulatory properties point to roles beyond the mere salvage of
extracellular nucleosides. A link between CNT2 function and energy metabolism has recently
been provided by the demonstration that CNT2-mediated uptake of adenosine into IEC-6 cells
activates AMP-dependent protein kinase (AMPK), thus promoting the phosphorylation of
downstream targets such as acetyl CoA carboxylase-1 (Aymerich, Foufelle, Ferré, Casado and
Pastor-Anglada, submitted). In turn, CNT2 may modulate extracellular adenosine levels, since it
shows the highest affinity for adenosine (apparent Km values of 8μM) [4]. Although adenosine
also binds to CNT1 with high affinity [12], adenosine transport via CNT1 is either very low or
negligible [12, 13]. CNT2 co-localizes with the adenosine receptor A1R at the plasma membrane
of Fao cells, whereas the A1R protein has been localized at the basolateral domain of the
hepatocyte, as shown for CNT2 [8]. Moreover, CNT2-related transport activity is rapidly
increased after A1R agonist activation in rat hepatocytes and hepatoma cells, by a mechanism
that requires functional KATP channels [14], but does not seem to be associated with changes in
CNT2 abundance at the plasma membrane (preliminary observations).
Nevertheless, the occurrence of CNT2 protein in intracellular stores ([8], this study)
prompted us to examine whether CNT2 activity might be regulated by changes in its subcellular
distribution in rat liver parenchymal cells. Among a panel of putative modulators, bile acids were
chosen since they regulate nucleoside metabolism in regenerating rat liver, where CNT2-related
Biochemical Journal Immediate Publication. Published on 3 Jan 2006 as manuscript BJ20051232
Copyright 2006 Biochemical Society

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transport activity is up-regulated [10]. In particular taurocholate promotes either nucleoside
catabolism or incorporation into DNA, depending on nutritional status [15, 16]. Moreover, bile
acids are bioactive molecules. They are physiological ligands for the farnesoid X receptor [17,
18] and they transcriptionally modulate enzymes of their own metabolism [19-21] as well as the
basolateral Na-dependent bile acid cotransporter NTCP [22]. Bile acids also promote the
translocation of their own export pump BSEP into the canalicular membrane [23]. Furthermore,
bile acid transport from the sinusoid to the canalicular domain of the hepatocyte requires the
polarized localization of particular transporter proteins, whose expression and activity are also
dependent on the differentiated status of hepatocytes as well as on bile acids themselves [24] (for
a recent review see [25]). Here it is reported that bile acids rapidly increase CNT2-related
transport activity by changing its subcellular localization, thus increasing expression at the
plasma membrane. This mechanism is sensitive to the inhibition of the PI3kinase/Extracellular
Regulated Kinase (ERK) transduction signal pathway, but independent of the KATP-mediated
activation of CNT2 previously described. More interestingly, this effect is novel to bile acid
physiology, and provides the first evidence of short-term regulated translocation of CNT2
into/from the plasma membrane.
Biochemical Journal Immediate Publication. Published on 3 Jan 2006 as manuscript BJ20051232
Copyright 2006 Biochemical Society

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MATERIALS AND METHODS

Chemicals and reagents
Colchicine, deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), taurocholic
acid (TCA), ursodeoxycholic acid (UDCA) dibutyryl AMPc, cycloheximide and the inhibitor of
tyrosine kinase genisteine were purchased from Sigma (Saint Louis, Missouri). The selective and
cell-permeable inhibitor of MAP kinase (MEK) PD 98059 and the selective and irreversible
inhibitor of phosphatidyl-inositol-3 kinase (PI3K) wortmannin were from Calbiochem (La Jolla,
Calif.).
3H-guanosine was from Moravek (CA, USA) and (γ-32P) ATP from Amersham
Biosciences (Buckinghamshire, UK). Proteina A-agarosa was from Roche Molecular
Biochemicals (Mannheim, Germany). All other reagents were of analytical grade.

Fao and HepG2 cells, isolated rat hepatocytes, and culture conditions
The rat hepatoma cell line Fao (purchased from ACACC) was routinely cultured in
Coon´s F12 medium supplemented with 10% (v/v) calf serum, 2 mM glutamine and a mixture of
antibiotics (100 u/ml penicillin G, 0.1 mg/ml streptomycin and 0.25 μg/ml fungizone). This cell
line, which is derived from the H4IIEC3 cell line Reuber H35, is well differentiated, expresses
numerous liver-specific enzymes and transcription factors [26] and retains significant CNT2
activity and expression [9].
The human hepatocyte carcinoma cell line HepG2 (ECACC) was cultured in DMEM
supplemented with 10% (v/v) calf serum, 2 mM glutamine and a mixture of antibiotics.
All animals used in this study were maintained, handled and killed according to the
European Union laws on biomedical research involving laboratory animals. Hepatocytes from
adult rats were isolated from male Wistar rats by the classical anterograde collagenase perfusion
method as described elsewhere [27]. Isolated hepatocytes were then seeded in Earle’s E-199
medium supplemented with 2% (v/v) fetal calf serum and a mixture of antibiotics (100 u/ml
penicillin G, 0.1 mg/ml streptomycin and 0.25 μg/ml fungizone).
Primary cultures were analyzed 15 h after seeding, and Fao cells were used at 60-70%
confluence. Before monitoring the effects of bile acids on nucleoside transport activity and
carrier expression, primary cultures and cell lines were incubated overnight in a serum-free BSA-
supplemented medium.
Biochemical Journal Immediate Publication. Published on 3 Jan 2006 as manuscript BJ20051232
Copyright 2006 Biochemical Society