Regulation of Plasma Membrane Localization of the Na + -taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

Background: Little is known about the effect of hyperosmotic hepatocyte taurocholate polypeptide Results: Hyperosmolarity induces a Fyn-dependent retrieval of Ntcp, which is reversed by tauroursodeoxycholate via a β 1 -integrin-mediated formation of cAMP. Conclusion: Fyn and β 1 -integrins are novel regulators of Ntcp localization. Significance: The study provides new insights into the regulation of bile salt transport. ABSTRACT In perfused rat liver, hepatocyte shrinkage induces a Fyn-dependent retrieval of the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane (1) leading to cholestasis. However little is known about the effects of hyperosmolarity on short-term regulation of the Na + -taurocholate cotransporting polypeptide (Ntcp), the major bile salt uptake system at

induced Fyn activation and triggered re-insertion of the hyperosmotically retrieved Ntcp into the membrane.
This was associated with dephosphorylation of the Ntcp on serine residues. Insertion of Ntcp by TUDC was sensitive to the integrin inhibitory hexapeptide GRGDSP and inhibition of protein kinase A. TUDC also reversed the hyperosmolarity-induced retrieval of Bsep from the canalicular membrane. These findings suggest a coordinated, oxidative stressand Fyn-dependent retrieval of sinusoidal and canalicular bile salt transport systems from the corresponding membranes. Ntcp insertion was also identified as a novel target of β 1 -integrindependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.
Disturbances of transcellular transport of bile acids in hepatocytes can result in the accumulation of potentially toxic bile acids inside the hepatocyte and can lead to cell damage and liver dysfunction (2)(3)(4)(5)(6). It is therefore critical that hepatocellular uptake and secretion of bile acids are coordinately regulated. At the canalicular membrane bile salt excretion is mediated by the bile salt export pump (Bsep) (7) and to a lesser extent by the multidrug resistance-associated protein 2 (Mrp2) (8). The expression of Bsep and Mrp2 in the canalicular membrane of the hepatocyte is regulated by oxidative stress (9)(10)(11), lipopolysaccharide (LPS) (12)(13)(14), drugs (15,16) and ambient osmolarity (1,10,(17)(18)(19). The cellular hydration state is a dynamic parameter that can change physiologically within minutes depending on the ambient osmolarity, nutrient supply, influence of hormones and oxidative stress. Alterations in the cellular hydration represent an important mechanism for the metabolic control and a messenger linking cell function to hormonal and environmental alterations.
The Na + -taurocholate cotransporting polypeptide (Ntcp) (30) is the major uptake system for conjugated bile salts from the blood into liver parenchymal cells. Loss of Ntcp results in increased plasma levels of conjugated bile acids, as also confirmed by a recent report on human NTCP deficiency (31), which is clinically characterized by mild hypotonia, growth retardation and delayed motor milestones. Functional studies show that the underlying mutation results in a markedly reduced uptake activity of taurocholate (TC), which is due to an absence of the mutant protein in the plasma membrane of the hepatocyte (31). Recent studies suggest that the cholestatic bile salt taurolithocholate (TLC) inhibits hepatic TC uptake and decreases plasma membrane level of Ntcp (32). Also taurochenodeoxycholate (TCDC) inhibits TC uptake at the sinusoidal membrane in a chelerythrine-and cypermethrin-dependent way (33). Ntcp is a serine/threonine phosphorylated protein (34) and underlies complex regulation (reviewed in Ref. 35). cAMP can induce an increase in TC uptake by insertion of Ntcp into the plasma membrane (36) via increases in intracellular Ca 2+ and subsequent activation of protein phosphatase 2B (PP2B) via Ca 2+calmodulin kinase (reviewed in Ref. 35). This is associated with a dephosphorylation of Ntcp at Ser-226 (37). Hepatocyte swelling stimulates rapid Ntcp insertion into the basolateral membrane and increases TC uptake in hepatocytes (38), but nothing is known about a regulation of Ntcp by hyperosmotic cell shrinkage and the hydrophilic, non-toxic bile acid TUDC.
The hydrophilic bile acid ursodeoxycholic acid (UDCA), which in vivo is rapidly converted to its taurine conjugate TUDC (39), is a mainstay in the treatment of cholestatic liver disease, such as primary biliary cirrhosis or intrahepatic cholestasis of pregnancy (40)(41)(42)(43)(44). Intrahepatic cholestasis results in an accumulation of potentially toxic bile acids inside the hepatocyte, that can cause hepatocyte death due to activation of CD95-dependent apoptosis (45,46). TUDC has potent anticholestatic and antiapoptotic properties. Experimental and clinical evidence suggests that at least three mechanisms are responsible for the hepatoprotective properties of TUDC in cholestatic disorders (for a review see 47): (1) stimulation of hepatobiliary secretion, (2) cytoprotection of hepatocytes against bile acidinduced apoptosis and cytokine-induced injury, and (3) protection of cholangiocytes against cytotoxicity of hydrophobic bile acids. We could recently demonstrate that both, the choleretic and antiapoptotic effects of TUDC are mediated by an activation of α 5 β 1 -integrins (48,49). TUDCinduced integrin activation leads to the formation of cAMP, which triggers inactivation of the CD95 and induction of a MAP kinase phosphatase (49). The stimulation of biliary excretion is also triggered via TUDC-induced integrin activation and subsequent activation of the mitogenactivated protein kinases p38 MAPK and Erks (19,50). This dual MAP kinase activation results in a TUDC-induced stimulation of biliary excretion by rapid targeting and insertion of intracellularly stored transport ATPases, such as Bsep or Mrp2 into the canalicular membrane (25,29). However, nothing is known about the effects of TUDC on the localisation of the basolateral Ntcp, the major uptake system for conjugated bile acids into the hepatocyte. The present study shows that TUDC triggers the insertion of the bile salt transporter Ntcp into the plasma membrane and that integrins are the sensor for this mechanism. Our study also shows that hyperosmotic hepatocyte shrinkage triggers an oxidative stress-, protein kinase Cζ-and Fyndependent internalization of Ntcp, which is reversed by TUDC in a β 1 -integrin-and cAMPdependent manner. The findings are not only of interest for the understanding of hepatocellular bile acid handling and TUDC action in liver, but may also be relevant for hepatitis B virus (HBV) entry into the hepatocyte, which was recently shown to be mediated by Ntcp (51). Indeed, recent data suggest that interleukin-6 inhibits HBV entry into the hepatocyte through downregulation of NTCP (52).
Liver perfusion -Livers from male Wistar rats (140-160 g) were perfused in a non-recirculating manner as described previously (54). As a perfusion medium the bicarbonate-buffered Krebs-Henseleit saline plus L-lactate (2.1 mmol/l) and pyruvate (0.3 mmol/l) gassed with 5% CO 2 and 95% O 2 at 37°C was used (305 mosmol/l, normoosmotic). Hyperosmotic (385 mosmol/l) perfusion was performed by raising the NaCl concentration in the medium. Addition of inhibitors, Db-cAMP and TUDC to the influent perfusate was made by dissolution into the Krebs-Henseleit buffer. Viability of the perfused livers was assessed by measuring lactate dehydrogenase leakage into the perfusate. The portal pressure, the effluent K + concentration and pH were continuously monitored. Livers from male control mice (wild type, 8-10 weeks; on C57BL/6 background), or p47 phox knockout mice (8-10 weeks, C57BL/6J-Ncf1m1J/J, The Jackson Laboratory, Maine, USA) were perfused in an adapted version.
Preparation and culture of primary rat hepatocytes -Hepatocytes were isolated from livers of male Wistar rats (160-180 g) by a collagenase perfusion technique in an adapted version as described previously (55). Animals were fed ad libitum with a standard diet. Aliquots of rat hepatocytes were plated on collagen-coated culture plates and maintained in bicarbonatebuffered Krebs-Henseleit medium (115 mmol/l NaCl, 25 mmol/l NaHCO 3 , 5.9 mmol/l KCl, 1.18 mmol/l MgCl 2 , 1.23 mmol/l NaH 2 PO 4 , 1.2 mmol/l Na 2 SO 4 , 1.25 mmol/l CaCl 2 ), supplemented with 6 mmol/l glucose in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. After 2 h, the medium was removed and the cells were washed twice. Subsequently the culture was continued for 24 h in William`s Medium E, supplemented with 2 mmol/l glutamine, 100 nmol/l insulin, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 100nmol/l dexamethasone and 5% FBS. After 24 h, experimental treatments were performed using William`s Medium E that contained 2 mmol/l glutamine and 100 nmol/l dexamethasone. The viability of hepatocytes was more than 95% as assessed by trypan blue exclusion. In order to allow comparisons to previous studies, in these cell culture experiments hyperosmolarity of 405 mosmol/l was used, whereas in rat liver perfusions we used 385 mosmol/l.
Densitometric fluorescence intensity analysis -Cryosections of perfused rat liver for the analysis at the basolateral membrane were stained for Ntcp and for Na + /K + -ATPase (plasma membrane marker protein). For negative controls, primary antibodies were omitted in each experiment. Na + /K + -ATPase profiles were selected according apparent integrity and comparability.
Acceptable Na + /K + -ATPase intensity profiles have a sufficiently high peak fluorescence in the central part (corresponding to the basolateral membrane) and low intracellular fluorescence. For analysis of the canalicular membrane, cryosections were stained for Bsep and the tight junction protein ZO-1, which forms the sealing border between canalicular and sinusoidal membrane. Apparent integrity and comparability of the canaliculi was assumed when the bordering tight junction lines (detected by the immunostained ZO-1) were intact, run in parallel, and showed a width of 1.12 to 1.82 µm. Densitometric analysis was performed as described previously (10,17). For analysis of digitalized microscopic pictures of the membranes, the software Image-Pro Plus (Media Cybernetics, Rockville, USA) was used. The profile of the fluorescence intensity was measured over a thick line at a right angle to the membrane. The length of the line was always 8 µm. The mean fluorescence intensity to each pixel over the line perpendicular to the length was calculated by Image-Pro Plus. The obtained data (pixel positions with the associated pixel intensities, red and green channel) were transferred to an MSexcel data sheet (Microsoft Excel for Windows, Redmond, USA). Each measurement was normalized to the sum of all intensities of the respective measurement. Values are given as means ± SEM. Densitometric analysis of protein distribution in immunofluorescence images were performed using Wilcoxon rank sum test. p < 0.05 was considered statistically significant. Data from at least 10 different areas per tissue sample and from at least 3 independent liver preparations were processed.
Immunoblot analysis -Liver samples were immediately lysed at 4°C by using a lysis buffer containing 20 mmol/l Tris-HCl (pH 7.4), 140 mmol/l NaCl, 10 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 1% (v/v) Triton X-100, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l sodium vanadate, 20 mmol/l β-glycerophosphate, protease inhibitor and phosphatase inhibitor cocktail. The lysates were kept on ice for 10 min and then centrifuged at 8000 rpm for 8 min at 4°C, and aliquots of the supernatant were taken for protein determination using the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany). Equal amounts of protein were subjected to sodium dodecyl sulfate/ polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes using a semidry transfer apparatus (GE Healthcare, Freiburg, Germany). Membranes were blocked for 60 min in 5% (w/v) bovine serum albumin containing 20 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, and 0.1% Tween 20 (TBS-T) and exposed to primary antibodies overnight at 4°C. After washing with TBS-T and incubation at room temperature for 2 h with horseradish peroxidasecoupled anti-mouse or anti-rabbit IgG antibody, respectively (all diluted 1:10000), the immunoblots were washed extensively and bands were visualized using the FluorChem E detection instrument from ProteinSimple (Santa Clara, CA). Semi-quantitative evaluation was carried out by densitometry using the Alpha View image acquisition and analysis software from ProteinSimple. Protein phosphorylation is given as the ratio of detected phospho-protein/total protein.
Immunoprecipitation -Liver samples were harvested in lysis buffer containing 136 mmol/l NaCl, 20 mmol/l Tris-HCl, 10% (v/v) glycerol, 2 mmol/l EDTA, 50 mmol/l β-glycerophosphate, 20 mmol/l sodium pyrophosphate, 0.2 mmol/l Pefablock, 5 mg/l aprotinin, 5 mg/l leupeptin, 4 mmol/l benzamidine, 1 mmol/l sodium vanadate, supplemented with 1% (v/v) Triton X-100. The protein amount was determined as described above. Samples containing equal protein amounts were incubated for 2 h at 4°C with the respective antibody in order to immunoprecipitate the required protein. Then protein A-/G-agarose (Santa Cruz Biotechnology, Heidelberg, Germany) was added and incubated at 4°C overnight. Immunoprecipitates were washed thrice with lysis buffer supplemented with 0.1% (v/v) Triton X-100 and then transferred to Western blot analysis as described above.
Detection of ROS -Primary hepatocytes were seeded on collagen-coated plates (BD Falcon, Heidelberg, Germany) and cultured for 24 h. Cells were incubated with PBS containing 5 µmol/l CM-H 2 DCFDA for 30 min at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air. To detect ROS generation, CM-H 2 DCFDA-loaded cells were supplemented again with culture medium and then exposed to the respective stimuli for the indicated time period. Thereafter cells were washed briefly using ice-cold PBS, and lysed in 0.1% Triton X-100 (v/v) dissolved in aqua bidest. Lysates were centrifuged immediately (10000 × g, 4°C, 1 min) and fluorescence of the supernatant was measured at 515-565 nm using a luminescence spectrometer LS-5B (PerkinElmer Life Sciences, Rodgau, Germany) with an excitation wavelength of 488 nm. Fluorescence intensity of controls was set to 1 and fluorescence found under the respective treatment is given relative to it.
Realtime-PCR analysis -Total RNA was isolated using the RNAeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturers´ protocol.
Statistical analysis -Results from at least 3 independent experiments are expressed as mean values ± SEM. n refers to the number of independent experiments. For each experimental treatment and time point analyzed, a separate control experiment was carried out. Differences between experimental groups were analyzed by student`s t test or one-way analysis of variance following Dunnett's multiple comparison post hoc test where appropriate (GraphPad Prism; GraphPad, La Jolla, USA; Microsoft Excel for Windows). p < 0.05 was considered statistically significant.

RESULTS
Hyperosmolarity triggers Ntcp retrieval from the basolateral membrane in the perfused rat liver -In order to study the subcellular localization of the bile salt transporter Ntcp in hepatocytes, immunofluorescence studies on cryosectioned tissues using confocal laserscanning microscopy were performed (Fig. 1). For labelling of the plasma membrane, liver sections were stained simultaneously with specific antibodies against the plasma membrane marker protein Na + /K + -ATPase and against Ntcp, respectively. The profile of the fluorescence intensity of Ntcp and Na + /K + -ATPase was measured over a thick line (8 µm) perpendicular to the membranes of adjacent hepatocytes. The mean fluorescence intensity of each pixel along this line was calculated by Image-Pro Plus and the obtained and normalized data were plotted on a line graph (Fig. 1). Subcellular Ntcp distribution in normoosmotically (305 mosmol/l) and hyperosmotically (385 mosmol/l)-perfused livers was quantified by densitometric fluorescence intensity analysis as described above. Within 30 min of hyperosmotic perfusion the fluorescence profile of Ntcp shifted to significantly lower peak values and became more broadened (p < 0.05, Fig. 2A), suggestive for an internalization of the Ntcp in response to hyperosmolarity. In contrast, no changes in Na + /K + -ATPase distribution were observed under these conditions ( Fig. 2A). To discriminate Ntcp distribution at a subcellular level, SR-SIM in Ntcp and Na + /K + -ATPase costained cryosections was performed. In SR-SIM images, obtained from hyperosmotically (385 mosmol/l)-perfused rat livers, intracellular vesicular Ntcp fluorescence intensity was markedly increased within 30 min and maintained for at least 60 min (Fig. 2B). Localization of the basolateral membrane protein Na + /K + -ATPase remained unchanged as compared to normoosmotic control conditions (Fig. 2B). These results suggest that hyperosmolarity induces within 30 min endocytosis of Ntcp in the intact rat liver, which lasts for at least 60 min. Hyperosmolarity-induced retrieval of Ntcp was not accompanied by changes in Ntcp mRNA expression during the first 60 min of hyperosmotic exposure (0.96 ± 0.07-fold, n.s., n = 4, Fig. 2C). However, a significant decrease of Ntcp mRNA levels was seen after treatment with hyperosmotic medium for 180 min (0.76 ± 0.04fold, p < 0.05, n = 4) or more (Fig. 2C).
Hyperosmolarity-induced retrieval of Ntcp from the basolateral membrane is mediated by activation of the Src family kinase Fyn -Hyperosmolarity was shown to trigger a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent formation of reactive oxygen species (ROS) (6,57) and to activate the Src family kinases Yes and Fyn, whereas no c-Src activation became detectable within 180 min of hyperosmotic exposure (1). Hyperosmotic Fyn and Yes activation were sensitive to inhibition by NAC, apocynin and SU6656, whereas PP-2 inhibited hyperosmotic Fyn, but not Yes activation (1). Ntcp distribution in hyperosmotically (385 mosmol/l)-perfused livers in absence and presence of inhibitors of the NADPH oxidase-driven ROS generation (apocynin and NAC) or of Src kinases (PP-2, SU6656) was quantified by densitometric fluorescence intensity analysis (Fig. 3). Fig. 3A depicts the perfusion plans chosen for these experiments. As shown in Fig. 3B-D, NAC (10 mmol/l) and apocynin (20 µmol/l) inhibited the hyperosmolarity (385 mosmol/l, t = 30 min)induced Ntcp retrieval from the basolateral membrane, whereas Na + /K + -ATPase distribution remained unchanged. These findings point to a role of NADPH oxidase-driven ROS formation in triggering transporter retrieval from the plasma membrane.
Also the Src kinase inhibitors SU6656 (1 µmol/l) and PP-2 (250 nmol/l) prevented hyperosmotic Ntcp retrieval from the basolateral membrane (Fig. 3D). Because PP-2 (58) inhibits Fyn but not Yes activation, whereas SU6656 (59) inhibits both, hyperosmotic Yes and Fyn activation, these findings suggest that Fyn is involved in the hyperosmotic retrieval of Ntcp from the basolateral membrane. The role of NADPH oxidase in triggering hyperosmotic Ntcp retrieval from the plasma membrane was also studied in livers from p47 phox knock-out mice. As shown in Fig. 4, hyperosmolarity induced within 30 min the internalization of Ntcp in livers from wild type mice, whereas no retrieval was detectable in livers from p47 phox knock-out mice.
TUDC reverses hyperosmolarity-induced internalization of Ntcp via integrin-dependent protein kinase A activation -The choleretic bile salt TUDC induces a rapid insertion of intracellularly stored Mrp2 and Bsep into the canalicular membrane of the hepatocyte (19,25), which increases the bile salt excretory capacity in a microtubule-dependent way (60,61). As shown by SR-SIM, TUDC (20 µmol/l, 30 min) was able to reverse the hyperosmolarity-induced internalization of Ntcp (Fig. 5). Because β1integrins were identified as sensors for TUDC in rat liver (19,48), the integrin-inhibitory peptide GRGDSP which binds to α 5 β 1 -integrin with a K D of 396 nM (62) was used to study the involvement of β1-integrins in TUDC-induced Ntcp insertion into the plasma membrane. GRGDSP (10 µmol/l) inhibited the counteracting effect of TUDC on hyperosmotic Ntcp retrieval, while the inactive control peptide GRADSP (10 µmol/l) was ineffective (Fig. 5A,C). As shown recently, TUDC activates via β1-integrins the protein kinase A (PKA) (49). When H89 (2 µmol/l), a PKAinhibitor, was perfused 30 min before and during the experiment, the effect of TUDC on hyperosmolarity-induced Ntcp retrieval was largely abolished, while Na + /K + -ATPase immunostaining remained unchanged under all conditions (Fig. 5C). Similar findings were obtained with Db-cAMP (50 µmol/l), which is known to increase the uptake capacity of TC by increasing plasma membrane content of Ntcp (36). This effect was also H89 (2 µmol/l)sensitive (data not shown). All inhibitors, i.e. GRADSP, GRGDSP and H89 had per se no effect on Ntcp localization (data not shown).
TUDC reverses the hyperosmolarityinduced internalization of the bile salt transporter Bsep -In line with a TUDC-induced reversal of hyperosmotic Fyn activation, TUDC not only prevented the hyperosmolarity-induced Ntcp retrieval from the plasma membrane, but also the hyperosmolarity-induced Bsep retrieval from the canalicular membrane (Fig. 9), which was previously shown to be Fyn-dependent, too (1). Fig. 9A shows the experimental approach, which was also used in previous studies (1,9,10,17). Immunofluorescence stainings of the canalicular bile salt transporter Bsep and of ZO-1 (a protein of the tight junction complex), were investigated by confocal laser scanning microscopy and densitometric analysis. ZO-1 delineates the bile canaliculi and a linear staining pattern of ZO-1 was found under all conditions. During normoosmotic perfusion (305 mosmol/l) Bsep was located predominantly in the canalicular membrane, i.e. between the two rows of ZO-1 fluorescence (Fig. 9A,B). Hyperosmotic perfusion (385 mosmol/l) led to an increase of Bsep fluorescence intensity inside the hepatocytes at the expense of its canalicular localization, suggesting Bsep internalization (Fig. 9B). Subsequent addition of TUDC (20 µmol/l) to the same liver preparation led to an almost complete disappearance of intracellular Bsep, indicating reinsertion of Bsep into the canalicular membrane. Under all these conditions ZO-1 fluorescence distribution remained unaffected (Fig. 9B).
Paralleling the inhibition of ROS formation, the hyperosmolarity-induced Fyn phosphorylation was sensitive to inhibition of PKCζ by the specific PKCζ pseudosubstrate, and by the broad spectrum PKC inhibitors chelerythrine and Gö 6850 (Fig. 11A). Gö 6850 also prevented the hyperosmolarity-induced retrieval of Ntcp (Fig. 11B). Gö 6976, a selective inhibitor of Ca 2+ -dependent PKC isoforms had no significant effect on hyperosmotic Fyn phosphorylation (Fig. 11A). These findings suggest that activation of PKCζ in response to hyperosmolarity may play a role in triggering hyperosmotic Fyn phosphorylation (Fig. 11A) and Ntcp retrieval (Fig. 11B).

DISCUSSION
Hyperosmolarity was shown to stimulate the retrieval of Bsep and Mrp2 from the canalicular membrane (1,10,17), whereas the hydrophilic bile acid TUDC stimulates the insertion of these transporters into the canalicular membrane (25,29). However, the effects of hyperosmolarity and TUDC on the short-term regulation of the basolateral transporter Ntcp remained unexplored. The present study shows that (i) hyperosmolarity induces an oxidative stress-and Fyn-dependent retrieval of Ntcp from the membrane, and (ii) that TUDC is able to stimulate re-insertion of Ntcp into the plasma membrane by a β 1 -integrin-and PKA-dependent mechanism. To what extent TUDC-induced PI3 kinase activation (64) contributes to this effect remains to be established. Fig. 12 schematically summarizes our current view on short-term regulation of the basolateral bile salt transporter Ntcp.
Under normoosmotic conditions, Ntcp was found mainly in the basolateral membrane and also in transporter-containing vesicles underneath the plasma membrane ( Fig. 2 and 5). Hyperosmolarity increased the fraction of Ntcp, which is located inside the hepatocytes ( Fig. 2 and  5). This shift in Ntcp localization occured within 30 min, persisted for at least 60 min and was reversible in response to TUDC (Fig. 5). Activation of the Src kinase Fyn was shown to occur in response to hyperosmotic cell shrinkage and to trigger the retrieval of Bsep and Mrp2 from the canalicular membrane (1). As shown in the present study, hyperosmotic Fyn activation also triggers Ntcp retrieval from the basolateral membrane (Fig. 3). PP-2, which blocks the hyperosmolarity-induced phosphorylation of Fyn (1), abolished internalization of Ntcp (Fig. 3). Likewise, inhibitors of the upstream events leading to Fyn activation, such as the Noxinhibitor apocynin or the antioxidant NAC, prevented transporter internalization (Fig. 3). Furthermore, Ntcp retrieval was inhibited in p47 phox knockout mice (Fig. 4), in which the hyperosmolarity-induced activation of Fyn is abolished (1). These data strongly suggest that Fyn mediates not only Bsep and Mrp2 retrieval from the canalicular membrane, but simultaneously also triggers the retrieval of Ntcp from the basolateral membrane. This suggests a coordinated regulation of sinusoidal bile salt uptake and canalicular export systems, which may help to avoid accumulation of potentially toxic bile acids in the hepatocyte. This view is also corroborated by the finding that TUDC and cAMP could reverse hyperosmolarity-induced Fyn activation ( Fig. 6 and 7), Ntcp retrieval from the plasma membrane (Fig. 5) and Bsep retrieval from the canalicular membrane (Fig. 9). Thus, TUDC exerts its choleretic effect by a coordinated insertion of both, Ntcp and Bsep into plasma and canalicular membrane, respectively. A recent study showed that vesicle motility and ATP production is transiently reduced in a variety of cell types including hepatocytes after treatment with hyperosmotic medium (65). Movement of dextran-loaded endosomes was tracked by livecell imaging. Because dextran is distributed to early, late and recycling endosomes this approach however, will not distinguish between differences in motility among these compartments. Whether the motility is restricted also in newly endocytosed vesicles remains therefore unclear (65).
Hyperosmotic perfusion of rat liver induced a NADPH-oxidase dependent oxidative stress response as an upstream signal for Fyn activation. Cell hydration and oxidative stress are apparently closely interlinked. On the one hand cell shrinkage induces oxidative stress (66) and on the other oxidative stress leads to hepatocyte shrinkage (67). Inflammation is frequently accompanied by ROS formation and can induce cholestasis. It is likely that inflammation also triggers Ntcp retrieval from the plasma membrane. Indeed, proapoptotic bile salts such as taurolithocholic acid-3 sulfate or glycochenodeoxycholate which induce a NOX2-dependent ROS formation and cell shrinkage (68) also trigger Fyn activation and Ntcp internalization.
Hyperosmolarity-induced retrieval of Ntcp was not accompanied by a downregulation of Ntcp mRNA levels within 60 min, whereas LPS triggered both, Ntcp retrieval and a significant decrease of Ntcp expression at the mRNA and protein level (69). This difference between hyperosmotic and LPS-induced Ntcp retrieval may be explained by cytokine effects.
PKCs belong to a family of serine/threonine kinases which consists of at least 12 isoforms (reviewed in Ref. 70). These kinases are involved in the regulation of diverse cellular functions (reviewed in Ref. 70) including bile formation (reviewed in Refs. 71 and 35). It was suggested that choleretic and cholestatic effects may involve different PKC isoforms; however, the picture is not entirely consistent. The toxic bile acid TCDC inhibits bile salt transport across the sinusoidal membrane by PKC-and PP2Bmediated retrieval of Ntcp from the plasma membrane (33). Phorbol ester-induced endocytosis of Ntcp was shown to be in part mediated by Ca 2+ -dependent PKCs (56). Furthermore it was shown recently that PKCα can directly interact with Ntcp in stably Ntcptransfected HepG2 cells (72). PKCζ (73,74) was suggested to mediate a choleretic effect by inserting Ntcp into the membrane, whereas PKCε (75,76) may trigger cholestasis by retrieving Mrp2 from the canalicular membrane. However, PKCζ was also shown to trigger p47 phox phosphorylation and to induce oxidative stress (57) as upstream event for Bsep and Mrp2 retrieval from the canalicular membrane (1). The present study shows, that PKCζ is involved in the Fyn-regulated retrieval of the Ntcp from the membrane (Fig. 11), whereas PKCζ inhibitors were reported to prevent the cAMP-induced Ntcp translocation to the plasma membrane (73). These discrepant findings strongly suggest that regulation of Ntcp retrieval/insertion by PKCζ may dependent on the signalling context and the underlying stimulus.
As part of its choleretic effect, TUDC stimulates insertion of the canalicular transporter Bsep and Mrp2 by activation of Erks and p38 MAPK (25,50) and counteracts internalization in TLCinduced cholestasis (29). In the present study, the choleretic effect of TUDC may reside in the prevention of hyperosmotic Fyn activation and cAMP formation. Recently, β 1 -integrins were identified as sensors for TUDC in the liver (19, 48) and molecular dynamics simulation studies showed that TUDC directly interacts with α 5 β 1integrins resulting in an activation of β 1 -integrin and the initiation of the downstream integrin signaling (19,48). This also explains the TUDCinduced activation of c-Src as observed in Fig. 6C. Translocation of Ntcp to the plasma membrane by TUDC was sensitive to GRGDSP and H89 (Fig. 5), which suggests that also Ntcp insertion by TUDC involves a β 1 -integrin-mediated activation of PKA. Similarly to TUDC, cAMP significantly increased Ntcp translocation to the basolateral membrane in a PKA-dependent way (data not shown). The effect of cAMP is known to be mediated via two major pathways. Activation of PI3K, probably via PKA (77), leads to activation of Akt (78) and PKC isoforms (73,74,79), which in turn induce the translocation of Ntcp from an intracellular compartment to the membrane.
Ntcp is a serine/threonine phosphorylated protein (34) and dephosphorylation at Ser-226 (37) is associated with translocation to the plasma membrane. cAMP activates the serine and threonine protein phosphatase PP2B by increasing intracellular Ca 2+ . Activated PP2B directly dephosphorylates Ntcp allowing translocation to the basolateral membrane (80). It was shown recently, that TUDC by itself can induce a β 1integrin-dependent rapid generation of cAMP (49), which may explain Ntcp dephosphorylation in response to TUDC.
Taken together, the present study for the first time shows that hyperosmolarity induces the retrieval of Ntcp from the plasma membrane and that this effect is mediated by the Src family kinase Fyn which is activated by a PKCζdependent and NADPH-oxidase-driven ROS formation. TUDC counteracts the effect of hyperosmolarity through activation of a β 1integrin/PKA-signaling pathway, which prevents hyperosmotic ROS formation and Fyn activation and is associated with dephosphorylation of the Ntcp on serine residues.