Ursodeoxycholic acid stimulates the formation of the bile canalicular network

Biochemical Pharmacology 84 (2012) 925–935

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Ursodeoxycholic acid stimulates the formation of the bile canalicular network Yuki Ikebuchi 1, Hidetoshi Shimizu 1, Kousei Ito *, Takashi Yoshikado, Yoshihide Yamanashi, Tappei Takada, Hiroshi Suzuki Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 June 2012 Accepted 9 July 2012 Available online 20 July 2012

Ursodeoxycholic acid (UDCA) is a hepatoprotective bile acid used in the treatment of chronic liver diseases. Although several pharmacological effects, including choleresis and inhibition of apoptosis, have been proposed, the impact of UDCA on hepatic structure is not well understood. Here, the influence of UDCA on bile canalicular (BC) morphology was evaluated in vitro in immortalized rat hepatocytes (McA-RH 7777 cells) and primary rat hepatocytes. Cells cultured for 3 days in the presence of UDCA, the BC lumen was enlarged and the bile canaliculi were surrounded by multiple cells (5) with a continuous canal-like structure, reminiscent of the in vivo BC network. The effects were dependent on p38MAPK and conventional PKC in McA-RH cells, and partially dependent on p38MAPK, MAPK/ERK kinase, and conventional PKC in primary rat hepatocytes. These findings were then studied in vivo in a rat model of dimethylnitrosamine-induced hepatic injury, in which the BC network is significantly disrupted. In accordance with the in vitro observations, administration of UDCA (40 mg/kg/day) to the injured rats for 18 days improved the BC network compared with the vehicle control. Serum hepatic markers were not altered by UDCA treatment, suggesting that the morphological effects were due to the direct actions of UDCA on network formation. Our data provide new evidence of the pharmacological potential of UDCA in accelerating or regenerating BC network formation in vitro, in hepatic cell culture models, and in vivo in a rat model of hepatic injury, and provide a basis for understanding its hepatoprotective effects. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Ursodeoxycholic acid Bile canalicular Biliary excretion Bile acid

1. Introduction Bile acids are amphipathic compounds synthesized in the liver from cholesterol and then actively secreted into bile [1]. They play important roles in extracting phospholipids and cholesterol from the canalicular membrane of hepatocytes and thus promote the formation of mixed micelles in bile. Bile acids also participate in the solubilization of lipophilic compounds, such as dietary lipids, vitamins, and drugs, in the lumen of the small intestine [1,2]. Based on their biosynthetic host, bile acids are divided into two groups [1]. Primary bile acids are synthesized from cholesterol by hepatocytes, while secondary bile acids are synthesized from primary bile acids by the intestinal flora. In humans, the major primary bile acids are cholic acid (CA) and chenodeoxycholic acid (CDCA), and the major secondary bile acids are deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA). DCA and LCA are synthesized by the 7a-dehydroxylation of CA and CDCA, respectively. UDCA is synthesized by the epimerization of

* Corresponding author. Tel.: +81 3 5800 9192; fax: +81 3 5800 9442. E-mail address: [email protected] (K. Ito). 1 These authors contributed equally to this work. 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.07.008

7a-OH from CDCA. These five bile acids (CA, CDCA, DCA, UDCA, and LCA) are coupled with glycine or taurine to form conjugated bile acids. Thus, in humans, there are at least 15 different bile acids, 5 unconjugated and 10 conjugated [1]. UDCA is the active ingredient of a drug prepared from a crude zooid extract. Its use in the treatment of jaundice can be documented as far back as the Tang Dynasty in China. Although UDCA is the major bile acid in the black bear, it accounts for only 3% of the total bile acids in humans. Based on the exceptionally low detergent toxicity of UDCA compared with other bile acids, it has been used therapeutically in patients with bile duct disease, cholestasis, and gall stones [1]. Recent indications include the treatment of primary biliary cirrhosis and chronic hepatitis C. Moreover, the beneficial effects of UDCA in primary sclerosing cholangitis, intrahepatic cholestasis in pregnancy, and progressive familial intrahepatic cholestasis have been reported [1,3,4]. At least three mechanisms are believed to be involved in the pharmacological actions of UDCA [4]: (i) the protection of hepatocytes and cholangiocytes by the replacement of other hydrophobic, but toxic, bile acids; (ii) the inhibition of apoptosis induced by toxic bile acids; and (iii) the stimulation of biliary secretion by the up-regulation of apically inserted hepatic transporter proteins, including multidrug resistance-associated

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protein 2/ATP-binding cassette C family 2 (Mrp2) and bile salt export pump/ATP-binding cassette B family 11 (Bsep). The UDCAmediated stimulation of biliary secretion may in part be due to the acceleration of bicarbonate-chloride exchange by cholangiocytes [5,6]. In addition, UDCA is thought to be involved in modulating the immune response; for example, by inhibiting inflammatory cytokines [7,8] or by enhancing chemotaxis in inflammatory cells [9,10]. Considering its diverse clinical indications and benefits, multiple pharmacological mechanisms of action have been postulated for UDCA. Hepatocytes are polarized cells with distinct apical and basolateral membranes [11]. The apical membrane forms the lumenal space of the bile canaliculi (BC, also used herein as an abbreviation for ‘‘bile canaliculus’’ and ‘‘bile canicular’’). The BC lumens of adjacent hepatocytes are connected to each other, thereby forming a continuous tubular structure. Bile components are secreted into the BC by specialized efflux transporters [12] and are sequentially guided through the BC network to the intrahepatic and extrahepatic bile ducts surrounded by cholangiocytes [13]. The correct developmental formation of BC structures and the subsequent maintenance of the BC continuous network are indispensable to normal biliary secretion by the liver. Although one of the well-known clinical benefits of UDCA is its ability to improve biliary secretion, little is known about its effects on BC structure and morphology. Therefore, the aim of the present study was to investigate the UDCA response of BC network formation in vitro, in a rat hepatocyte model, and in vivo, in rats with chemicalinduced hepatic injury. The results showed, for the first time, that UDCA has a distinctive ability to accelerate BC network formation in cultured cells and to regenerate the BC network in liverdamaged adult rats.

2. Materials and methods 2.1. Materials UDCA was obtained from Wako (Osaka, Japan). Tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), and CA were obtained from Nacalai Tesque (Kyoto, Japan). Taurocholic acid (TCA), glycocholic acid (GCA), and Go¨6976, a conventional protein kinase C (PKC) inhibitor, were obtained from Sigma (St. Louis, MO). See Table 1 for more information on Go¨6976 and the other inhibitors listed in this section. PD98059, SB202190, and H-7 were obtained from Calbiochem (San Diego, CA); SB203580 and H-89 were obtained from Cayman Chemical (Ann Arbor, MI); SB202474 was obtained from Merck Ltd. (Tokyo, Japan); and KT5823 and Go¨6850 were obtained from Enzo Life Sciences (Plymouth Meeting, PA).

Table 1 Inhibitors used in the in vitro study. Inhibitor

Concentration (mM)

Target (reported IC50 or Ki values)

PD98050 SB202190 SB203580 SB202474 H-7

50 10 10 10 30

MAPK/ERK kinase (MEK) (IC50 = 2–7 mM) p38MAPK (IC50 = 50–100 nM) p38MAPK (IC50 = 600 nM) Inactive form of SB202190 and SB203580 PKC (Ki = 6.0 mM), PKG (Ki = 5.8 mM), PKA (Ki = 3 mM) PKCa, PKCbI, PKCbII, PKCg, PKCd, PKCe (IC50 = 10 nM) PKCa (IC50 = 2.3 nM), PKCbI (IC50 = 6.2 nM), PKCm (IC50 = 20 nM) PKG (IC50 = 234 nM) PKA (Ki = 48 nM)

Go¨6850

2

Go¨6976

2

KT5823 H-89

10 10

2.2. Cell culture McA-RH 7777 cells (McA-RH; Riken, Ibaraki, Japan) derived from a rat hepatoma were maintained at 37 8C in a humidified atmosphere supplemented with 5% CO2 in culture medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque), 10% fetal bovine serum (BioWest, Paris, France), and 1% penicillin– streptomycin (Nacalai Tesque). Rat Bsep cDNA inserted into the pEGFP-N1 plasmid (Clontech, Shiga, Japan) was transfected into McA-RH cells using FuGENE6 (Roche, Applied Science, Indianapolis, IN). G418 bisulfate (600 mg/ml, geneticin; Nacalai Tesque) selection of the transfected cells was carried out in the culture medium described above. Cells stably expressing enhanced green fluorescent protein-tagged Bsep (Bsep-GFP) were selected and maintained in culture medium containing 200 mg G418 bisulfate/ml. 2.3. Immunostaining McA-RH cells were pre-cultured for 72 h on sterilized glass coverslips in 6-well plates at a density of 1  105 cells/ml. The medium was then replaced with fresh medium containing UDCA or the other test compounds, and the cells were cultured for an additional 72 h, with fresh medium changes every 24 h. The cells were then washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 10 min, and then permeabilized for 5 min with PBS containing 0.1% Triton-X 100. After three more PBS washes, the specimens were blocked with PBS containing 1% bovine serum albumin (BSA) at room temperature for 30 min and then incubated with PBS containing 1% BSA and one of the following primary antibodies: anti-Mrp2 antiserum [14] or monoclonal antibody (clone M2III-6; Abcam, Cambridge, UK); anti-radixin antibody R21 [15] (Invitrogen, Carlsbad, CA). This step was followed by the incubation of the cells with the corresponding Alexa Fluor1-labeled secondary antibody (Invitrogen) or with phalloidin-TRITC (Invitrogen) to stain actin filaments (F-actin), or with Hoechst33342 dye (Sigma) to stain nuclei. The cells were viewed using laser scanning confocal microscopy and images were obtained with an FV1000 imaging system (Olympus, Tokyo, Japan). 2.4. Quantification of BC The staining properties of the BC indicators Bsep-GFP, Mrp2 (immunostained with anti-Mrp2 antiserum or antibody), and Factin (stained with phalloidin-TRITC) were first determined in control, untreated McA-RH cells (see Section 3, Fig. 1). The numbers of total BC (NtBC), extended globular BC (NExGBC, corresponding to a globular form of BC > 20 mm in diameter), and extended tubular BC (NExTBC, corresponding to an extended form of BC made up of several cells) were counted to obtain the extended BC (ExBC) formation ratio according to the formula: (NExGBC + NExTBC)/(NtBC). More than 300 BC (from a total of ten fields at 40 magnification) were counted in each sample, and the averages were calculated from at least three independent samples. 2.5. Construction of recombinant adenovirus The human Na+/TCA co-transporting polypeptide/solute carrier family 10A1 (NTCP) was amplified from total RNA isolated from HepG2 (liver hepatocellular carcinoma) cells. The cDNA was cloned first into the KpnI/XbaI site of the pcDNA3.1/mycHis vector (Invitrogen) and then into the SacII/NheI site of the pTRE-Shuttle2 vector (Adeno-X Tet-Off Expression System 1 Kit; Clontech/TakaraBio, Mountain View, CA). Recombinant adenoviruses were constructed according to the manufacturer’s protocol. For mock infections, recombinant adenoviruses containing enhanced GFP were similarly constructed. The Tet-regulatory adenoviruses

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Fig. 1. The effect of UDCA on BC morphology in McA-RH cells stably expressing Bsep-GFP. A, BC in the absence of UDCA; B, extended globular BC; and C, extended tubular BC in the presence of 200 mM UDCA for 72 h (see Section 2 for the experimental conditions). The cells were fixed and stained with phalloidin-TRITC (F-actin). Asterisks indicate the position of the BC lumen. Bar = 10 mm.

required for target protein expression were included in the kit. These recombinant adenoviruses were amplified in HEK293 cells and purified by cesium chloride gradient ultracentrifugation. Viral titers were determined using an Adeno-X Rapid Titer Kit (Clontech/ Takara-Bio). The adenovirus experiments described below were performed after the multiplicity of infection (MOI) had been optimized. Infections were carried out by seeding the cells onto glass coverslips and, 48 h later, adding adenoviruses to the McA-RH cell culture medium at a concentration of 10–20 MOI, followed by an additional 24 h of culture. The medium was then replaced with fresh medium containing UDCA or other bile acids, and the cells were cultured again, this time for 72 h. Expression of the recombinant or endogenous protein was confirmed by Western blotting using antic-myc antibody (A-14; Santa Cruz Biotechnology Inc., Heidelberg, Germany), anti-Bsep antiserum [16], anti-Mrp2 antiserum [14], or anti-a-tubulin antibody (ab15246; Abcam). 2.6. Uptake of bile acids by McA-RH cells McA-RH cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin–streptomycin for 48 h at a density of 1  105 cells/ml in 6-well tissue culture plates. Adenoviruses were added to the medium at a concentration of 20 MOI for 24 h. The medium was then replaced with fresh medium containing each bile acid at 200 mM, and the cells were cultured for an additional 24 h followed by two washes with PBS containing 0.05% Tween-20 and two more washes with PBS only. Bile acids were extracted from the cells as described previously [17], with minor modifications. Samples consisting of the cell pellet, 300 ml of ethanol, and 1 nmol of 23nordeoxycholic acid (Steraloids Inc., Newport, RI) as an internal standard were prepared and then subjected to sonication, followed by heating at 60 8C for 30 min and 100 8C for 3 min. After a centrifugation step at 1600  g for 10 min at 15 8C, the supernatants were collected and ethanol (300 ml) was added to obtain a precipitate. These samples were mixed vigorously, and the supernatants were collected by centrifugation as above. The extraction was repeated twice. The pooled extracts were vacuum-concentrated, dissolved in 1 ml of distilled water, and purified through an Oasis1 HLB cartridge (Waters, Milford, MA) according to the manufacturer’s instructions. Purified extracts were vacuum-concentrated and finally dissolved in 150 ml of methanol. Bile acids were separated using an Acquity ultra-performance liquid chromatography system

(Waters) and detected with a Quattro Premier XE quadrupole tandem mass spectrometer (Waters) equipped with an ESI probe in negative-ion mode. 2.7. Primary rat hepatocytes Rat hepatocytes were prepared by collagenase perfusion of the livers from 7-week-old male Wistar rats (Japan SLC, Shizuoka, Japan), as previously described [18]. Cell viability (>90%) was determined by trypan blue exclusion. The isolated rat hepatocytes were cultured (3  105 cells/ml) in Williams’ medium E supplemented with 10% fetal bovine serum, 1% PCSM, 2 mM L-glutamine (Nacalai Tesque), 250 nU insulin (Sigma)/ml, and 1 mM dexamethasone, and then seeded onto rigid-collagen (Cellmatrix1 Type I-C, Nitta gelatin, Osaka, Japan)-coated glass coverslips in 6-well tissue culture plates followed by a 4 h incubation in 5% CO2 at 37 8C. The medium was then exchanged and the incubation continued for 48 h. After the addition of 200 mM UDCA or TCA in 0.6% DMSO (final), the hepatocytes were cultured for an additional 72 h, with a medium change every 24 h. 2.8. In vivo model of rat liver injury The 7-week-old male Wistar rats used in this study were housed in temperature- and humidity-controlled animal cages with a 12 h dark/light cycle and free access to water and rat chow (MF; Oriental Yeast, Tokyo, Japan). The experimental design consisted of four groups of 3–4 rats each. Rats in group 1 received an intraperitoneal injection of saline three times per week (three consecutive daily injections followed by 4 days without injections) for 3 weeks, followed by the oral administration of water as vehicle every day for 18 days. Rats in group 2 received an intraperitoneal injection of saline for 3 weeks as described above, followed by the oral administration of UDCA (40 mg/kg body weight, as an aqueous suspension) for 18 days. Rats in group 3 received an intraperitoneal injection of dimethylnitrosamine (DMN; Wako) (10 mg/kg body weight, three consecutive daily injections and then 4 days without injections) for 3 weeks, followed by the oral administration of water for 18 days. Rats in group 4 received an intraperitoneal injection of DMN for 3 weeks as described above, followed by the oral administration of UDCA for 18 days. Serum was collected from the jugular vein at the beginning of each week. At the end of the

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Fig. 2. Quantitative analysis of the effect of UDCA on BC morphology in McA-RH cells. McA-RH cells stably expressing Bsep-GFP were cultured for 72 h, followed by the addition of various concentrations of UDCA and culture for an additional 72 h. BC were subsequently identified in fixed cells by Bsep-GFP fluorescence and classified according to the criteria described in Section 2. A, The ratio of ExBC to total BC was defined as the number of ExBC (extended globular plus extended tubular BC) divided by the number of total BC (ExBC/Total BC). B, The distribution of the number of cells participating in forming single BC is shown. C, The UDCA concentration-dependent induction of ExBC. D, The UDCA concentration-dependent effect on total BC number. Results are presented as the mean  SE (n = 3). *p < 0.05 and **p < 0.01, significantly different from the result(s) in the absence of UDCA.

experiment, the rats were anesthetized with an intraperitoneal injection of 1.25 g urethane (Sigma–Aldrich)/kg. The common bile duct was cannulated with a 0.2 mm polyethylene tube (Natsume, Tokyo, Japan). Bile specimens collected at 10 min intervals

between 10 and 70 min after cannulation were weighed and the biliary volume was determined, assuming a specific gravity of 1.0 g/ml. The concentrations of bile acid, cholesterol, and phospholipid in bile were determined enzymatically using kits

Fig. 3. Effect of UDCA treatment on the endogenous expression of Mrp2 and Bsep in McA-RH cells. McA-RH cells without or with stable transfection of rat Bsep (‘‘parent’’ or ‘‘rBsep’’, respectively) were cultured for 3 days then medium was changed with fresh one containing 200 mM UDCA or vehicle (‘‘vehicle’’ or ‘‘UDCA’’) and cultured for 72 h then expression of Mrp2 (A), Bsep (B) and ExBC formation (D) were examined. Crude membrane fractions were prepared [34] and subjected to Western blot analysis with anti-Mrp2 antiserum, anti-Bsep antiserum and anti-a-tubulin antibody. For immunofluorescence detection of Mrp2, cells were fixed and stained with phalloidin-TRITC (Factin) and anti-Mrp2 antiserum (C). Bar = 10 mm.

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from Wako. Finally, the rats were killed, at which time blood and liver samples were collected and processed for immunohistochemical analysis. All animals were treated humanely in accordance with the guidelines issued by the National Institutes of Health (NIH publication 86-23, revised 1985). 2.9. Liver immunohistochemistry Liver specimens were fixed, permeabilized with hexane cooled by acetone on dry ice, and immediately frozen in liquid nitrogen. The samples were stored at 80 8C until frozen sections (5 mm thick) were prepared. Fixed and permeabilized frozen liver sections were incubated with antibodies against Mrp2 and radixin, followed by the corresponding secondary antibodies labeled with Alexa Fluor1. The liver sections were observed using a laser scanning confocal microscope equipped with an FV1000 imaging system (Olympus).

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2.10. Statistical analysis The results were compared using an analysis of variance (ANOVA) between groups, followed by the Student–Newman– Keuls test to compare all pairs of means. Comparison of two groups only was performed using the unpaired two-tailed Student’s t-test. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. UDCA stimulates the formation of extended BC in McA-RH cells Among the untreated McA-RH cells, 15% formed globular BC, as determined by F-actin staining. The BC were approximately 10 mm in diameter and they were surrounded by two neighboring cells (Fig. 1A). In addition to these ‘‘normal’’ BC, McA-RH cells cultured

Fig. 4. Effect of UDCA and other bile acids on BC morphology in McA-RH cells. A, The ratio of ExBC (ExBC/Total BC) was calculated in McA-RH cells cultured for 72 h in the absence of any bile acids, followed by an additional 72 h of culture in the presence of bile acids (200 mM). Results are presented as the mean  SE (control and UDCA, n = 15; other bile acids, n = 3). **p < 0.01 vs. control. B, Expression of NTCP in McA-RH cells infected with adenoviruses. Total cell lysates were separated by SDS-PAGE, and NTCP was detected by Western blotting. C, Uptake of bile acids by McA-RH cells infected with adenoviruses. Intracellular bile acids were extracted, and the extent of uptake was assessed using the UPLC/ESI-MS system. Results are presented as the mean  SE (n = 4). **p < 0.01, significantly different from the GFP group. D, The ratio of ExBC to total BC (ExBC/Total BC) was determined in McA-RH cells infected with the NTCP-carrying adenovirus 24 h before the addition of bile acids (see Section 2). Cells were infected with a GFP-carrying adenovirus in parallel experiments. BC were identified by immunostaining for Mrp2. Results are presented as the mean  SE (n = 3). **p < 0.01 vs. the control group; yp < 0.05 and zp < 0.01 vs. the GFP-carrying adenovirus group.

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in the presence of 200 mM UDCA formed globular BC larger than 20–30 mm in diameter (extended globular BC) and tubular BC formed by more than three cells (extended tubular BC) (Fig. 1B and C). They are hereafter jointly referred to as extended BC (ExBC). The apical resident transporters Bsep and Mrp2 were localized along the ExBC, as was ZO-1, a molecular component of tight junctions (data not shown). 3.2. UDCA stimulates ExBC formation in a concentration-dependent manner The addition of 200 mM UDCA was found to cause a significant seven-fold increase in the ExBC formation ratio (Fig. 2A). In the absence of UDCA, >60% of the BC were formed by two cells, while in the presence of UDCA, there were fewer such BC and, instead, the number of cells forming a single BC increased (Fig. 2B). Given that cells above and below the microscopy observation plane could not be accurately counted, the number of cells involved in BC formation was likely to have been underestimated, especially for ExBC formed by more than five cells. Consequently, these BC were not characterized further but simply denoted as ‘‘5 cells’’ (Fig. 2B). To further confirm the UDCA concentration dependence of ExBC formation, McA-RH cells were cultured in the presence of 12.5–400 mM UDCA, and the ExBC formation ratio was calculated. As shown in Fig. 2C, UDCA caused a significant concentrationdependent increase in the ExBC formation ratio but had essentially no effect on the number of total BC (Fig. 2D). 3.3. ExBC formation induced by UDCA is not dependent on biliary efflux transporters To exclude the possibility that the observed effect is merely a secondary consequence of enhanced secretion of UDCA and/or organic anions into the closed lumenal space, we checked the expression status of Bsep (bile acid transpoter) and Mrp2 (organic anion transpoter) in McA-RH cells. As a result, Mrp2 (Fig. 3A) but not Bsep (Fig. 3B) was endogenously expressed in parental McA-RH cells, and UDCA does not affect these proteins expression and localization (Fig. 3C). In spite of the absence of Bsep and unchanged Mrp2 expression/localization in parental McA-RH cells, ExBC was similarly induced by UDCA (Fig. 3D). These results indicate that UDCA induces ExBC independent of these biliary efflux transporters.

the GFP control (Fig. 4C). As shown in Fig. 4D, both taurine- and glycine-conjugated forms of UDCA (TUDCA and GUDCA), but not those of CA (TCA and GCA), significantly induced ExBC formation in cells that expressed NTCP. 3.5. p38MAPK and conventional PKC are involved in UDCA-induced ExBC formation in McA-RH cells TUDCA exerts anti-cholestatic action via signaling kinases, including p38MAPK [21], ERK1/2 [22,23], and PKCa and PKA [21,24]. We used inhibitors for these pathways (Table 1) to evaluate whether they were involved in the formation of ExBC. As shown in Fig. 5, UDCA-induced ExBC formation was not affected by PD98059, indicating a minor, if any, contribution of the MEK pathway. Both SB202190 and SB203580, but not their inactive homologue SB202474, inhibited UDCA-induced ExBC formation to about 55% of the control, i.e., to the level in the absence of inhibitor. The tested concentrations of PD98059 and SB202190 were sufficient to completely inhibit the phorbol 12-myristate 13-acetate-induced activation of MEK and p38MAPK in McA-RH cells, respectively (data not shown), suggesting that p38MAPK but not MEK is involved in UDCA-induced ExBC formation in McA-RH cells. Moreover, the contribution of conventional PKC was demonstrated based on the following observations. Firstly, H-7 inhibited UDCA-induced ExBC formation to 35% of that determined in the absence of inhibitor. Secondly, Go¨6850 and Go¨6976 inhibited UDCA-induced ExBC formation to the same extent as H-7. Thirdly, both KT5823 and H-89 failed to inhibit UDCA-induced ExBC formation. The observed effects were not due to a non-specific cytotoxicity of the inhibitors because the cells remained viable during the incubation period (data not shown). Collectively, these results indicate that, in McA-RH cells, the p38MAPK and conventional PKC signaling pathways are at least partially involved in UDCA-induced ExBC formation. 3.6. p38MAPK, MEK, and conventional PKC are involved in UDCA- and TCA-induced ExBC formation in primary rat hepatocytes Since the above-described effects of UDCA were obtained in immortalized hepatocytes, we thought it important to confirm the

UDCA (200 μM) + PD98059

3.4. UDCA and its conjugates specifically stimulate ExBC formation In hepatocytes, UDCA is conjugated with taurine and glycine to form TUDCA and GUDCA, respectively. Since UDCA accounts for only 3% of the total bile acids in humans, whereas TCA and GCA are the major bile acids, we investigated the effect of TUDCA, GUDCA, and the other predominant human bile acids (TCA, GCA, and CA) on BC morphology. As shown in Fig. 4A, the induction of ExBC was observed only in the presence of UDCA. Conjugated bile acids are actively taken up by hepatocytes via the uptake transporter NTCP [19]. In the absence of NTCP, cellular uptake of conjugated bile acids is limited due to their hydrophilic nature. Since McA-RH cells do not express NTCP [20], it could not be ruled out that the conjugated bile acids examined in this study (TUDCA, GUDCA, TCA, and GCA) were unable to enter the cells and were thus prevented from exerting their effects. To evaluate this hypothesis, McA-RH cells were infected with an NTCP- or GFPcarrying adenovirus, and the ability of the conjugated bile acids to influence BC morphology was analyzed. NTCP protein expression was confirmed 4 days after adenoviral infection in the majority of the cells by immunostaining (data not shown) and was also readily detected by Western blot analysis (Fig. 4B), with a significant enhancement in the uptake of all bile acids tested compared with

* *

+ SB202190 + SB203580 + SB202474

** ** **

+ H-7 + Gö6850 + Gö6976 6 + KT5823 + H-89

**

+ SB202190 / H7 0

20 40 60 80 100 120 140 160 UDCA-induced ExBC (% of UDCA only)

Fig. 5. Signaling pathways involved in the UDCA-induced BC morphological changes. The ratio of ExBC to total BC (ExBC/Total BC) was calculated in McA-RH cells cultured for 72 h in the presence of 200 mM UDCA with or without the inhibitors listed in Table 1, which also shows their applied concentrations and other pertinent details. BC were identified by immunostaining with Mrp2 antibodies. The fraction of UDCA-induced ExBC was calculated by subtracting the ExBC formation ratio in cells treated with vehicle only (0.6% DMSO) from that in cells treated with 200 mM UDCA, and was set as 100% ‘‘UDCA (200 mM)’’. Results are presented as the mean  SE (n = 3). *p < 0.05 and **p < 0.01 vs. ‘‘UDCA (200 mM)’’.

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results in primary rat hepatocytes. As in McA-RH cells, large globular or tubular BC were observed in primary rat hepatocytes after 3 days of culture in medium containing 200 mM UDCA (Fig. 6B and C). The ratio of ExBC to total BC (Fig. 6D) and the number of cells forming a single BC (Fig. 6E) increased in the presence of UDCA. These results indicated that the induction of ExBC by UDCA is not limited to immortalized hepatocytes but also occurs in primary hepatocytes. Furthermore, although TCA did not have any effect in McA-RH cells, it induced ExBC formation in primary rat hepatocytes (Fig. 7A). Examination of the inhibitor sensitivity of UDCA-induced ExBC formation showed significant effects for both PD98059 and SB202190, suggesting the involvement of the MEK and p38MAPK pathways, respectively. In addition, UDCA-induced ExBC formation was significantly inhibited by H-7, as well as by Go¨6850 and Go¨6976. The simultaneous application of all three inhibitors (PD98059, SB202190, and H-7) further potentiated the inhibitory effect compared to either single applications or combinations of any two of these agents (Fig. 7B). Similar inhibitor sensitivities were obtained for TCA-induced ExBC formation (Fig. 7C). Collectively, these results show that in primary rat hepatocytes, the kinases p38MAPK, MEK, and conventional PKC are involved in the ExBC formation induced by UDCA and TCA.

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3.7. Establishment of an in vivo rat liver injury model To extend our findings in vivo, the effect of UDCA on BC morphology in a rat model of liver injury was studied. In nonperturbed rats, the BC network is well organized and multiple hepatocytes participate in forming a continuous network structure. Therefore, to mimic chronic liver disease and the therapeutic effects of prolonged UDCA administration in humans, we chose to test our in vitro findings in a rat model of chronic liver injury characterized by a disrupted BC structure, specifically asking whether UDCA was able to induce the regeneration of the BC network. The liver injury model was based on DMN, a highly toxic organic chemical that covalently binds to crucial biological molecules, including nucleic acids and proteins, and causes the metabolic activation of microsomes. When administered repeatedly to rats, DMN leads to chronic liver injury accompanied by liver fibrosis and, ultimately, to hepatocellular necrosis [25]. Since the effect of DMN on BC structure had not been previously examined, we began this in vivo study by characterizing liver enzyme levels, sclerosis, histology, fibrosis, and BC network structure in the DMNinjured liver. Rats administered DMN for 3 weeks showed a gradual decrease in weight gain and an increase in serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total

Fig. 6. The effect of UDCA on BC morphology in primary rat hepatocytes. The cells were pre-cultured for 48 h, followed by the addition of 200 mM UDCA in DMSO (final 0.6%) and an additional 72 h of culture. The cells were fixed and then immunostained with radixin antibody (green) and phalloidin-TRITC (red). A, Normal BC in control, untreated cells; B, extended globular BC; and C, extended tubular BC in the presence of 200 mM UDCA. Asterisks indicate the position of the BC lumen. D, The ratio of ExBC to total BC was defined as the number of ExBC (extended globular BC and extended tubular BC) divided by the number of total BC (ExBC/Total BC). E, The distribution of the number of cells participating in the formation of single BC. Results are presented as the mean  SE (n = 6); **p < 0.01 vs. 0 mM. Bar = 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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A vehicle

***

UDCA (200 μM)

**

C ((200 μM) TCA 0

***

0.10 0.20 ExBC / Total BC

B

C

UDCA (200 μM) μ

TCA (200 μM) μ

** *** *** *** ***

+ PD98059 + SB202190 + H-7 + Gö6850 + Gö6976

+ SB202190 + H-7 + Gö6850 + Gö6976

+ KT5823

+ KT5823

+ H-89

+ H-89

***

+ PD / SB

*** ***

+ PD / H-7 7 + SB / H-7

***

+ PD / SB / H-7 0

###

20 40 60 80 100 120

** * ** * **

+ PD98059

*** ** ***

+ PD / SB + PD / H-7 7 + SB / H-7

***

+ PD / SB / H-7 0

#

20 40 60 80 100 120 TCA-induced ExBC A only) (% of TC TCA

UDCA-induced ExBC (% of UDC A only) UDCA l )

Fig. 7. Signaling pathways involved in bile acid-induced BC morphological changes in primary rat hepatocytes. The cells were pre-cultured for 48 h, followed by the addition of 200 mM UDCA or TCA in DMSO (final 0.6%) and an additional 72 h of culture in the presence or absence of the inhibitors listed in Table 1, which also shows their applied concentrations and other pertinent details. The cells were fixed and then immunostained with radixin antibody and phalloidin-TRITC. (A) The ratio of ExBC to total BC was significantly increased in the presence of 200 mM UDCA or TCA. The fractions of (B) UDCA-induced and (C) TCA-induced ExBC were calculated by subtracting the ExBC formation ratio in cells treated with vehicle only (0.6% DMSO) from the ratio in cells treated with 200 mM UDCA or 200 mM TCA, with the latter values defined as 100%, respectively. Results are presented as the mean  SE (n = 3). (A) **p < 0.01 and ***p < 0.001; (B) **p < 0.01 and ***p < 0.001 vs. ‘‘UDCA (200 mM)’’; ###p < 0.001 vs. ‘‘PD/SB’’, ‘‘SB/H7’’, and ‘‘PD/H-7’’; (C) *p < 0.05, **p < 0.01, and ***p < 0.001 vs. ‘‘TCA (200 mM)’’; #p < 0.05 vs. ‘‘PD/SB’’, ‘‘SB/H-7’’, and ‘‘PD/H-7’’. ANOVA followed by a Student-Newman-Keuls test.

bilirubin compared with the saline-treated control group (Fig. 8A). Pronounced superficial sclerosis of the liver, disruption of liver histology, and an increased area of fibrosis were also observed, as evidenced by Azan and hematoxylin and eosin staining (data not shown). Finally, BC immunostaining for the markers radixin and Mrp2 showed a regular and continuous network in the livers of control, saline-treated rats but a severely disrupted one in the livers of DMN-treated rats (Fig. 8B). These results confirmed that the repeated administration of DMN induces liver injury accompanied by fibrosis and a degenerated BC structure. 3.8. UDCA improves BC structure in vivo UDCA (40 mg/kg/day) or water was administered daily for 18 days to rats with established liver injury and body weight, and the levels of serum markers were continuously monitored (Fig. 8A). At the end of the treatment period, the recovery of BC structure (Fig. 9) and biliary excretion functions (Table 2) were examined in detail. In the control (saline-vehicle and saline-UDCA) animals with no exposure to DMN, the BC network was regular and continuous, while in DMN-treated rats that did not receive UDCA (DMNvehicle), it was irregular and discontinuous (Fig. 9). However, in DMN-treated rats subsequently administered UDCA (DMN-UDCA), the appearance of the BC was less irregular and more continuous (Fig. 9) compared with that of the DMN-vehicle group. In contrast, there was little difference between the DMNvehicle and DMN-UDCA groups with respect to body weight change, ALT, ALP, and total bilirubin (Fig. 8A), nor did the four groups significantly differ in terms of liver weight, bile flow, and

biliary components (total bile acids, cholesterol, phospholipid) (Table 2). These findings showed that after the cessation of DMN intoxication DMN-induced liver injury spontaneously improved, whereas in the absence of UDCA treatment, the recovery of canalicular network integrity was significantly delayed. 4. Discussion This study showed that exogenously administered UDCA stimulates the formation of a continuous BC structure in vitro, in McA-RH cells and primary rat hepatocytes, and in vivo in a rat model of chronic liver injury. Not only UDCA but also TUDCA and GUDCA significantly induced ExBC formation in McA-RH cells overexpressing NTCP (Fig. 4D), whereas this was not observed for TCA, GCA, and CA, even though the cellular uptake of all bile acids tested increased dramatically (Fig. 4C and D). These results suggested that TUDCA and GUDCA, but not TCA, GCA, and CA, are the active compounds taken up by McA-RH cells. Interestingly, CDCA (7a-OH type), a stereoisomer of UDCA (7b-OH type), also failed to induce ExBC formation (data not shown). Thus, there may be an intracellular target that is both specific for the UDCA moiety and transmits the signal resulting in the induction of ExBC in McARH cells. This putative target does not seem to recognize CA, TCA, GCA, or CDCA. Previous reports of a role for the kinases p38MAPK [21], ERK1/2 [22,23], and PKC and PKA [21,24] in the various pharmacological actions of TUDCA and its conjugates also suggested the activity of a similar signaling pathway in ExBC formation. Indeed, we were able to show the involvement of p38MAPK and conventional PKC in UDCA-induced ExBC formation in McA-RH cells. Although further

Y. Ikebuchi et al. / Biochemical Pharmacology 84 (2012) 925–935

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Fig. 8. DMN-induced body weight changes, elevation of serum markers, and evidence of a disrupted BC network. A, The time profile of the changes in body weight and serum markers during treatment with DMN or saline for the first 3 weeks followed by UDCA or vehicle for the last 18 days (n = 3–4). B, The rats were sacrificed at day 21 and frozen liver sections (5 mm thick) were immunostained with radixin (green) and Mrp2 (red) antibodies. Bar = 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

analysis is needed to elucidate the components and mechanism of this signaling network, our data support the hypothesis that UDCA (and possibly other bile acids, including TCA) makes use of these multiple signaling pathways to induce BC extension. Recently, Fu et al. showed that the in vitro induction of BC network extension by TCA, TCDCA, and TUDCA (but not UDCA) was dependent upon a pathway comprising cAMP-Epac-MEK-LKB1AMPK [26,27]. The importance of LKB1 was also demonstrated in vivo in mice lacking hepatic LKB1 [28]. Our results in McA-RH cells differed from those reported by Fu et al. [27] in several aspects. In that study, TCA but not UDCA induced BC extension, with the MEK inhibitor PD98059 completely suppressing the response to TCA in sandwich-cultured primary rat hepatocytes. However, we showed that, in McA-RH cells, UDCA but not TCA was able to induce ExBC and that MEK inhibition did not inhibit UDCA-induced ExBC formation. Although there is no obvious explanation for the discrepancies regarding UDCA, TCA, and MEK, they point to the presence of several distinct signaling pathways by which bile acids stimulate extended BC formation, with the relative contribution of each one depending on the cell type and the culture conditions. Support for this hypothesis comes from our finding that TCAinduced ExBC formation in primary rat hepatocytes was MEKdependent (Fig. 7), consistent with the results of Fu et al. [27], whereas this was not observed in McA-RH cells (Fig. 4). However,

our results in primary rat hepatocytes still differ in several respects from those of Fu et al. [27], i.e., the positive response to UDCA, and the additional contributions of the p38MAPK- and conventionalPKC-mediated pathways in our study (Fig. 7). Again, the relative contributions of the various signaling pathways leading to the stimulation of ExBC formation by bile acids may be specific to the cell type, such as primary vs. immortalized (e.g., McA-RH cells) hepatocytes, and to the culture conditions, i.e., collagen sandwich [26–28] vs. non-sandwich cultures (present study). In fact, in a previous study, the sensitivity of the MEK pathway in primary rat hepatocytes was shown to depend on the culture conditions. Specifically, p42/p44, also known as ERK1/2 (downstream phosphorylation substrate of MEK1/2), is phosphorylated by dexamethason treatment under non-sandwich culture condition, while that is de-phosphorylated by the same treatment under sandwich culture condition. No alteration was observed for p38MAPK pathway irrespective of the culture conditions [29]. In DMN-injured rats, the disrupted structural continuity of the BC was significantly improved by UDCA treatment (Fig. 9). Since UDCA has multiple functions, such as cytoprotection, the inhibition of apoptosis, and the stimulation of biliary secretion, it is possible that the recovery of BC structure in vivo was part of an overall recovery of cellular function. However, serum markers (ALT, ALP, total bilirubin), liver histology (hematoxylin and eosin

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Y. Ikebuchi et al. / Biochemical Pharmacology 84 (2012) 925–935

Fig. 9. UDCA accelerates the recovery from DMN-induced BC disruption in vivo. Frozen liver sections from rats treated with 3 weeks saline + 18 days vehicle (saline-vehicle), 3 weeks saline + 18 days UDCA (saline-UDCA), 3 weeks DMN + 18 days vehicle (DMN-vehicle; spontaneous recovery group), or 3 weeks DMN + 18 days UDCA (DMN-UDCA; treatment group). The rats were sacrificed at day 39 and frozen liver sections (5 mm thick) were immunostained with radixin (green) and Mrp2 (red) antibodies. Bar = 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

staining), and fibrosis (Azan staining) (data not shown) were not responsive to UDCA treatment (compare DMN-UDCA rats with the DMN-vehicle group). Hence, the accelerated recovery of the BC network in our rat model can be attributed to a distinct pharmacological action of UDCA rather than to its indirect effects, such as protection from hepatocellular injury. Similarly of interest was the fact that UDCA minimally affected BC structure in non-DMN-treated rats (Fig. 9, saline-UDCA). It may be that damaged hepatocytes somehow acquire a higher sensitivity to UDCA than their healthy counterparts. The dependency of the cellular response to UDCA on cell type and culture condition, as discussed above, lends relate to this hypothesis. It is reported that proliferation of hepatocytes, rather than cholangiocytes, is selectively induced by DMN treatment [30]. That is one of the reasons why we choose DMN model in vivo and focus on hepatocytes in vitro. Alternatively, proliferation of cholangiocytes,

rather than hepatocytes, is selectively induced by bile duct ligation (BDL). Interestingly, ERK is significantly activated in proliferating cholangiocytes by common BDL [30] and UDCA further increased cell proliferative response induced by partial BDL [31]. These observations agree well with the idea that UDCA has also something to do with the regulation of cholangiocytes via ERK under certain liver damaged condition. Impact of UDCA on the intrahepatic bile duct morphology needs further investigation using in vitro cholangiocytes culture models [32,33] as well as appropriate hepatic injury model such as partial BDL rat model [31] in the future study. Accordingly, in the clinical setting, the pharmacological effect of UDCA and the relative contribution of these mechanisms would differ depending on the disease and/or the response of the individual patient, as previously suggested [5]. This remains an important point to be clarified in the future as it would improve the efficacy of UDCA therapy in patients.

Table 2 Bile constituents at the conclusion of the in vivo experiment. Rats were treated as described in Fig. 8, and bile samples obtained from rats in each group on day 39, i.e., at the conclusion of the in vivo experiment (3 weeks + 18 days), were analyzed.

Liver weight (g/kg body weight) Bile flow (ml/min/g liver) Bile acids (mM) Cholesterol (mg/dl) Phospholipid (mg/dl)

Saline-vehicle

Saline-UDCA

DMN-vehicle

DMN-UDCA

(n = 4)

(n = 4)

(n = 3)

(n = 3)

6.18  0.49 1.27  0.04 21.9  5.2 10.6  2.5 319  46

6.30  0.39 1.30  0.05 21.5  1.0 11.0  3.1 430  28

5.63  0.38 1.16  0.01 17.8  3.1 5.4  1.9 395  47

5.55  0.28 1.33  0.16 20.3  2.8 8.0  3.1 416  132

Y. Ikebuchi et al. / Biochemical Pharmacology 84 (2012) 925–935

In conclusion, our results provide evidence for the pharmacological potential of UDCA and its conjugates to accelerate or recover BC network formation in cultured hepatocytes and in adult rats, respectively. Although the direct molecular target of UDCA remains to be identified, our results indicate that these bile acids exert their functions intracellularly via multiple specific signal pathways. Acknowledgement This work was supported by the Ministry of Education, Science and Culture of Japan [Grand-in-Aid 17081006 and 12021149 for scientific research on Priority Areas]. References [1] Hofmann AF. The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 1999;159:2647–58. [2] Oude Elferink RP, Paulusma CC, Groen AK. Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases. Gastroenterology 2006;130:908–25. [3] Lindor KD, Kowdley KV, Luketic VA, Harrison ME, McCashland T, Befeler AS, et al. High-dose ursodeoxycholic acid for the treatment of primary sclerosing cholangitis. Hepatology 2009;50:808–14. [4] Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited. Hepatology 2002;36: 525–31. [5] Beuers U. Drug insight: mechanisms and sites of action of ursodeoxycholic acid in cholestasis. Nat Clin Pract Gastroenterol Hepatol 2006;3:318–28. [6] Renner EL, Lake JR, Cragoe Jr EJ, Van Dyke RW, Scharschmidt BF. Ursodeoxycholic acid choleresis: relationship to biliary HCO-3 and effects of Na+–H+ exchange inhibitors. Am J Physiol 1988;254:G232–41. [7] Invernizzi P, Salzman AL, Szabo C, Ueta I, O’Connor M, Setchell KD. Ursodeoxycholate inhibits induction of NOS in human intestinal epithelial cells and in vivo. Am J Physiol 1997;273:G131–8. [8] Yoshikawa M, Tsujii T, Matsumura K, Yamao J, Matsumura Y, Kubo R, et al. Immunomodulatory effects of ursodeoxycholic acid on immune responses. Hepatology 1992;16:358–64. [9] Liu L, Sakaguchi T, Cui X, Shirai Y, Nishimaki T, Hatakeyama K. Liver regeneration enhanced by orally administered ursodesoxycholic acid is mediated by immunosuppression in partially hepatectomized rats. Am J Chin Med 2002;30:119–26. [10] Santoro P, Raimondi F, Annunziata S, Paludetto R, Annella T, Ciccimarra F. Unconjugated bile acids modulate adult and neonatal neutrophil chemotaxis induced in vitro by N-formyl-met-leu-phe-peptide. Pediatr Res 2002;51:392–6. [11] Zegers MM, Hoekstra D. Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells. Biochem J 1998;336(Pt 2):257–69. [12] Jonker JW, Stedman CA, Liddle C, Downes M. Hepatobiliary ABC transporters: physiology, regulation and implications for disease. Front Biosci 2009;14: 4904–20. [13] Strazzabosco M, Fabris L. Functional anatomy of normal bile ducts. Anat Rec (Hoboken) 2008;291:653–60. [14] Ninomiya M, Ito K, Horie T. Functional analysis of dog multidrug resistanceassociated protein 2 (Mrp2) in comparison with rat Mrp2. Drug Metab Dispos 2005;33:225–32.

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