- Email: [email protected]
taurocholate cotransporting polypeptide during pregnancy in the rat
Journal of Hepatology 38 (2003) 148–155 www.elsevier.com/locate/jhep
Down-regulation of the Na 1/taurocholate cotransporting polypeptide during pregnancy in the rat Marco Arrese 1,*, Michael Trauner 2, Meenakshisundaram Ananthanarayanan 3, Margarita Pizarro 1, Nancy Solı´s 1, Luigi Accatino 1, Carol Soroka 4,5, James L. Boyer 4,5, Saul J. Karpen 6, Juan Francisco Miquel 1, Frederick J. Suchy 3 1 Department of Gastroenterology, Pontificia Universidad Cato´lica de Chile, School of Medicine, Marcoleta # 367, Santiago 6510260, Chile Division of Gastroenterology and Hepatology, Department of Internal Medicine, Karl-Franzens University School of Medicine, Graz, Austria 3 Department of Pediatrics, Laboratory of Developmental and Molecular Hepatology, Mount Sinai School of Medicine, New York, NY 10029, USA 4 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA 5 Liver Center, Yale University School of Medicine, New Haven, CT, USA 6 Texas Children’s Liver Center, Department of Pediatrics/Division of GI and Nutrition, Baylor College of Medicine, Houston, TX, USA 2
Background: Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents. Aim: To assess the expression and function of the main BA importer, the Na 1/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats. Methods: BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation. Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence. The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1-a (HNF1-a), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays. Results: A significantly reduced BA uptake and decreased Ntcp mRNA levels (240%) and protein mass (260%) was observed in pregnant rats. Nuclear extracts from pregnant rats showed a marked decrease of HNF1-a and RXR:RAR binding activities by 280 and 240% of basal activity, respectively. In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats. Conclusions: Pregnancy is associated with reduced Ntcp expression and function in the rat. Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Bile acid transport; Transporters; Na 1/taurocholate cotransporting polypeptide
1. Introduction
Received 24 April 2002; received in revised form 18 October 2002; accepted 22 October 2002 * Corresponding author. Tel.: 156-2-6863-820; fax: 156-2-6397-780. E-mail address: [email protected] (M. Arrese). Abbreviations: BA, bile acids; BSP, bromosulphthalein; Ntcp, Na 1/taurocholate cotransporting polypeptide; Oatp-1, organic anion transporting polypeptide-1; Bsep, bile salt export pump; Mrp2, multidrug resistant-associated protein; HNF1-a: hepatocyte nuclear factor 1-a; RXR:RAR, retinoid X receptor:retinoid acid receptor; Stat5, signal transducer and activator of transcription 5; EMSA, electrophoretic mobility shift assays.
Pregnancy is a physiological state known to affect bile secretory function [1]. Reduction of bile acid (BA) and bilirubin uptake as well as retention of the organic anion bromosulphthalein (BSP) have been reported in pregnant animals and similar changes have been postulated to occur in humans whose sera contain high levels of estrogens and progesterone during gestation [2,3]. These changes may have clinical implications with regard to the occurrence of clinical cholestasis during pregnancy [4].
0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S 0168 -82 78(02)00379-3
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
The recent cloning of a number of membrane transporters in hepatocytes have led to new insights into how cholephilic compounds are transported from blood to bile. Specifically, the molecular identity of transport systems involved in the transport of BA and organic anions has been elucidated [5]. Uptake of conjugated BA at the basolateral plasma membrane of the hepatocytes occurs mainly by a sodiumdependent mechanism mediated by the Na 1/taurocholate cotransporting polypeptide [Ntcp, Slc10a1]. Sodium-independent transport of non-conjugated bile acids also occurs at the basolateral membrane of hepatocytes although its physiological relevance is less clear [5]. This type of transport is thought to be mediated by members of the family of multispecific organic anion transporting polypeptides (Oatp’s) that transport unconjugated BA in a sodium-independent manner [6]. After intracellular transport, BA are excreted at the canalicular membrane of the hepatocyte mainly by an ATP-dependent mechanism that is mediated by the bile salt export pump (Bsep, Abcb11, [7]). Non-BA organic anions are mainly transported into bile by another canalicular transporter, the Multidrug resistant-associated protein 2 (Mrp2, Abcc2) that has an important function in the biliary excretion of endogenous metabolites, such as glucuronosyl-bilirubin, as well as many exogenous compounds [5,8]. Studies assessing the expression and function of hepatocyte membrane transporters during experimental conditions such as obstructive and hepatocellular cholestasis have consistently found that sinusoidal transporters such as Ntcp and Oatp-1 (Slc21a1) are downregulated during cholestatic injury [8,9]. The canalicular export pump Mrp2 is also dowregulated in various models of experimental cholestasis [9]. In contrast, changes in the canalicular BA transporter Bsep are less intense and the expression of this transporter is relatively preserved in experimental models of cholestasis including estrogen-induced cholestasis [9,10]. The present study was conducted to study the effect of pregnancy on the expression of transporters involved in hepatic BA handling by the hepatocyte in the rat in order to determine the underlying molecular mechanism of altered BA transport during this physiological state [2,3]. We also assessed the expression of the canalicular export pump Mrp2 since organic anion excretion is also affected by pregnancy in mammals [1,2]. Data presented in this communication show a significant down-regulation of the main sinusoidal BA transporter Ntcp, while Oatp-1 and Bsep expression remained unchanged. Ntcp down-regulation correlated with a significant decrease of staining in immunofluorescence studies and with a moderate decrease of BA uptake in isolated hepatocytes. Assessment of the nuclear transcription factors involved in the regulation of Ntcp [11,12] showed a significant decrease of binding activities of two critical nuclear transcription factors suggesting that pregnancy alters the transcription of Ntcp gene. Finally and in agreement with recent data [13], we found a decreased protein expression of Mrp2 in pregnant rats. Thus, down-regulation of Ntcp and
149
Mrp2 may explain some of the observed changes of bile secretory function during pregnancy in rodents 2. Material and methods 2.1. Chemicals Sodium salt of taurocholate (TC) was purchased from Sigma Chemical Company, (St. Louis, MO). [ 3H]TC (2.1 Ci/mmol) was obtained from DuPont NEN w Research Products (Boston, MA). [ 32P] deoxycytidine triphosphate was obtained from Amersham, Arlington Heights, IL. All other chemicals were of highest purity commercially available.
2.2. Animals Experiments were conducted in timed pregnant Sprague–Dawley rats weighing 210–230 g. Two time points of pregnancy were used: day 15 (PD15) and day 20 of pregnancy (PD20). The Scientific Advisory committee of the Catholic University of Chile School of Medicine approved the study protocols. Animals received humane care in compliance with the National Research Council’s criteria as outlined in Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985). Animals were anesthetized with a single dose of sodium pentobarbital (50 mg/kg body wt, intraperitoneally) and the liver removed after a short perfusion with saline solution.
2.3. Transport studies TC transport studies were performed using isolated, short-term cultured hepatocytes as described by Simon et al. [14] with minor modifications.
Fig. 1. Protein mass of hepatic bile acid transporters in male, nonpregnant female (NP) and pregnant (day 20 of pregnancy, PD20) rats. Membrane fractions were isolated from control rats and pregnant rats and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 50 mg protein/lane), and subsequently transferred to nitrocellulose membranes as described in Section 2. A representative Western blot is shown. Experiments were performed at least twice for each transporter. Molecular weights are given in kilodaltons (kDa). Densitometric analysis of protein levels showed that pregnancy is associated with a significant reduction in protein mass of Ntcp in PD20 rats while no change was seen in either Oatp-1 and Bsep protein expression (control [NP] ¼ 259 764 ^ 35 410 arbitrary densitometric units versus PD20 ¼ 77 013 ^ 9778 arbitrary densitometric units, P , 0.005). In addition, a selective decrease in Ntcp protein mass was observed in non-pregnant female rats when compared to male rats confirming previous observations on the existence of gender differences in Ntcp expression and function [21].
150
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Maximum transport of the dye BSP was performed in intact rat as previously described [15] to assess Mrp2 function.
sequence [6] and polyclonal anti-rat Mrp2 [17]. Immunoreactive bands were quantified by laser densitometry.
2.4. Tissue immunofluorescence
2.6. Messenger RNA (mRNA) studies
Qualitative distribution of Ntcp was assessed by indirect immunofluorescence as described previously [16,17]. Digital images were recorded and processed using Adobe Photoshop (Adobe Systems Inc., San Jose, CA).
Total RNA was isolated from whole-liver tissue by acid guanidinium thiocyanate-phenol chloroform extraction [20]. Poly(A)* RNA was isolated using oligo(dT)n linked to magnetic Streptavidin beads with the PolyATtract mRNA Isolation System IV (Promega, Madison, WI). Northern blots were performed using complementary DNA probes for Ntcp, Oatp-1, Bsep and Mrp2 as previously described [16,17]. The blots were exposed to Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and signals were quantitated using ImageQuant e software. Differences in loading were corrected after reprobing the stripped blots for glyceraldehyde-3phosphate dehydrogenase (GAPDH). The size of mRNA was estimated by comparison to a 0.24–9.5 Kb RNA ladder (GibcoBRL, Gaithersburg, MD).
2.5. Western analysis The protein mass of hepatic transporters in liver tissue from control and pregnant rats groups was measured as previously described [11,17] using either membrane-rich microsomal fractions, to assess the protein expression of Ntcp and Oatp-1 or canalicular-enriched liver plasma membrane fractions obtained by the method of Rosario et al. [18] to assess Bsep and Mrp2 protein mass. Additionally, Mrp2 protein expression was assessed in both plasma membrane fractions and liver homogenates as recently described by Cao et al. [13]. Western analysis was performed using standard techniques using the Renaissance Western blotting kit (New England Nuclear, Boston, MA). Membranes were probed with an anti-Ntcp fusion protein IgG as previously described [11,19], polyclonal anti-rat Oatp-1, polyclonal anti-rat Bsep antibody against a C-terminus 13-amino acid
2.7. Preparation of nuclei and nuclear protein extraction Liver nuclei were prepared from normal and PD20 animals and extracted as described previously [11]. Nuclear protein yields were similar in controls and pregnant animals.
Fig. 2. Protein mass and messenger RNA levels of Ntcp in control and pregnant rats at two time points (PD15 and PD20) during gestation: Membrane fractions were isolated from control (NP, n ¼ 3) and pregnant female rats [day 15 (PD15) and 20 (PD20) of pregnancy] and subjected to SDS-PAGE (100 mg protein/lane), and subsequently transferred to PVDF transfer membranes as described in Section 2. (A) Upper panel: Representative immunoblot. Autoradiographs of three independent samples are shown for each group. Molecular weights are given in kilo Daltons (kDa). Lower panel: Densitometric analysis of protein levels. Autoradiographs were quantified by laser densitometry, and data (mean ^ SD) are expressed as percentage of controls. *P , 0.0001 compared with controls. (B) Total mRNA was isolated as described in Section 2 and Northern blotting was performed using a radiolabelled probe for Ntcp as described in Section 2. Oatp-1 message was also quantitated for comparison. Upper panel: Representative Northern blot. Autoradiographs of three independent samples are shown for group. Each lane contains 30 mg of total RNA. Blots were stripped and reprobed for GAPDH to confirm equal loading and RNA integrity. Message sizes are given in kilobases (kb). The experiments were repeated twice. Lower panel: Quantitative analysis of Ntcp and Oatp-1 steady state RNA levels. Blots were quantified by PhosphorImager and normalized individually to GAPDH expression. Data (mean ^ SD) are expressed as Ntcp or Oatp-1/GAPDH ratio in arbitrary densitometric units. All the experiments were repeated at least twice.
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 3. Protein expression and function of the multidrug resistanceassociated protein 2 (Mrp2) in control and pregnant rats. Top: Total liver homogenate from control non-pregnant (NP) and 20 days-pregnant rats (PD20) were prepared according Cao et al. [13] and examined for the protein mass of Mrp2, the main non-bile acid organic anion canalicular transporter. Representative immunoblot showing a 50% decrease in protein mass of Mrp2 in pregnant rats compared with controls (NP: 628 353 ^ 12 099 arbitrary densitometric units in NP versus 315 444 ^ 4348 in PD20, n ¼ 3 in each group, P , 0.02). Bottom: Hepatobiliary transport of the model Mrp2 substrate BSP in NP and PD20 rats. BSP was infused through the jugular vein in a stepwiseincreasing rates as described [15]. Maximum secretory rate was calculated as the mean of the three highest consecutive values of BSP secretion and expressed as micrograms of BSP per minute per gram of liver. A significant decrease in BSP was observed in pregnant rats compared with controls (29.4 ^ 4.6 versus 19.2 ^ 1.4, in NP (n ¼ 5) and PD20 (n ¼ 6) rats, respectively, P , 0.05).
2.8. Electrophoretic gel mobility shift assays A total of 2–10 mg of nuclear extracts were incubated on ice for 30 min with 2 £ 10 4 cpm of 32P end-labeled oligonucleotide as described previously [11]. Specific oligonucleotides (sense strands sequences with their position in rat ntcp promoter are shown in Fig. 5) were used in electrophoretic mobility shift assays (EMSA) to assess the binding activities of transcription factors. Double-stranded oligonucleotide probes were endlabeled and purified according to standard procedures. In competition assays, 100-fold molar excess of the specific unlabeled oligonucleotide was added to the binding mixtures along with the labeled oligonucleotide.
151
tic transporters at the protein level, immunoblotting was performed in microsomal-enriched (analysis of Ntcp and Oatp-1) and canalicular membrane fractions (analysis of Bsep and Mrp2). Representative Western blots are shown in Figs. 1–3. Pregnancy was associated with a significant reduction in Ntcp protein mass in PD15 and PD20 rats while no change was seen in either Oatp-1 or Bsep protein expression. In addition, a selective decrease in Ntcp protein mass was also observed in non-pregnant females when compared to male rats. This confirms previous observations showing that Ntcp is dimorphically expressed in rat liver due to gender differences in transcription with males exhibiting greater amounts of Ntcp [21]. Analysis of Mrp2 protein expression showed a decrease in the protein mass of this transporter in agreement with data from Cao et al. [13]. Although we consistently reproduced this result using both liver homogenates and plasma membrane fractions, best quality Western blots were obtained with liver homogenates prepared following the methods reported by Cao et al. [13]. Therefore, representative blots of Mrp2 are shown in Fig. 3. 3.2. Tissue immunofluorescence Qualitative distribution of hepatic transporters was studied by indirect immunofluorescence in control and pregnant rats (PD20). Fig. 4 shows representative images of immunostaining with Ntcp antibody demonstrating a decreased labeling of the sinusoidal membrane in livers from PD20 rats. In addition, and in agreement with data from Cao et al. [13], a decreased immunostaining for Mrp2 was also found in pregnant rats. 3.3. mRNA studies Steady state mRNA levels for Ntcp, Oatp-1, Bsep and Mrp2 were assessed by Northern blot analysis. Densitometric analysis using the Storm 860 Phosphorimager apparatus showed a significant reduction of liver Ntcp mRNA levels by approximately 40% in both PD15 and PD20 rats, when compared to non-pregnant controls (Fig. 2B). No changes were seen in steady state mRNA levels for Oatp1, one of the Na 1-independent basolateral organic anion transporters or the canalicular ATP-dependent transporters Bsep and Mrp2 (data not shown) at both time points studied.
2.9. Statistics 3.4. Transport studies All results are expressed as mean ^ SE. A two-tailed non-paired Student’s t-test was used to compare differences between groups. Values were considered significantly different when the P value was equal to or less than 0.05.
3. Results 3.1. Western analysis To determine if pregnancy affects the expression of hepa-
In order to investigate if pregnancy is associated with a reduced function of Ntcp, [ 3H]TC uptake was assessed in short-term cultured hepatocytes from control and PD20 rats. Sodium-dependent [ 3H]TC uptake was reduced by 38% in pregnant animals as compared to non-pregnant rats (322.66 ^ 60 pmol mg protein 1 30 s 21 in controls versus 200.04 ^ 15 pmol mg protein 21 30 s 21 in PD20 rats, mean ^ SE n ¼ 3 in each group, t-statistic 3.4, P , 0:05) while sodium independent uptake was essentially
152
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 4. Indirect immunofluorescent localization of Ntcp and Mrp2 in control and pregnant rats. Frozen liver sections from control non-pregnant (NP) and 20 days-pregnant rats (PD20) were used to assess qualitative distribution of Ntcp and Mrp2 by indirect immunofluorescence as described previously [16,17]. A decreased labeling of both sinusoidal and canalicular membrane was observed accounting for reduction in Ntcp and Mrp2 protein expression in pregnant rats. Superimposition of images is shown on the right.
unchanged (71.3 ^ 8.3 pmol mg protein 1 30 s 1 in controls versus 75.37 ^ 15.6 pmol mg protein 1 30 s 1 in PD20 rats, mean ^ SE n ¼ 3 in each group, P . 0:05). In addition, decreased expression of Mrp2 during pregnancy was associated to a reduced maximum transport of the dye BSP in pregnant rats (Fig. 3).
the three oligonucleotide probes, the specificity of the DNAprotein interactions was demonstrated by appropriate competition assays (Fig. 5).
3.5. Binding activities of critical transcription factors of Ntcp promoter
Ntcp is the major bile acid uptake system in the basolateral membrane of rat hepatocytes [5]. The expression of Ntcp gene is significantly down-regulated in obstructive as well as hepatocellular cholestasis (including estrogeninduced) in rats [9,14,16]. The aim of this study was to determine if the expression of the major BA transport proteins were altered during pregnancy, since reduced BA transport has been reported during this physiological state in rodents [22,23]. A significant down-regulation of the protein and mRNA levels of the principal BA importer, Ntcp was seen during late pregnancy, while no changes were seen in the expression of another sinusoidal transporter, Oatp-1 nor the canalicular BA export pump Bsep. Our data suggest that the decrease of Ntcp is related to a reduced activity of two critical transcriptional regulators of the Ntcp promoter (see Fig. 5). Ntcp down-regulation during pregnancy provides a molecular mechanism for the reduced BA uptake occurring in the pregnant rat [23] corresponding to increased levels of serum BA seen during pregnancy in humans [24]. In addition to Ntcp down-regulation, we also found a decrease in the protein expression and function of the main canalicular organic bile acid transporter Mrp2, in agreement with recently published data [13]. Since the original aim of this
Multiple elements in the rat Ntcp promoter are bound by positive-acting transcription factors: an hepatocyte nuclear factor 1-a (HNF1-a) site at 27/18, a retinoic acid-response element (250/237), and tandem signal transducer and activator of transcription 5 (Stat5) sites (2936/2928 and 2912/ 2904). EMSA were performed using nuclear extracts from PD20 rats in order to address whether Ntcp down-regulation observed during pregnancy was related to decreased binding activities of some of these transactivators of transcription in the Ntcp promoter as previously reported by us in the endotoxin-induced cholestasis model [9,11]. Fig. 5 shows results of these experiments. A marked decrease of HNF1-a and retinoid X receptor:retinoid acid receptor (RXR:RAR) binding activities was found (277 and 260% compared to basal levels respectively, P , 0:01). In contrast, binding activity of Stat-5 was significantly increased by 150% in nuclear extracts from pregnant rats. These findings indicate that nuclei prepared from pregnant rats contain selectively decreased amounts of active HNF1-a and RXR:RAR levels and increased amounts of Stat5 transcription factor. For all
4. Discussion
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 5. Reduced binding activities of transcriptional regulators of Ntcp promoter during pregnancy. Hepatic nuclear extracts were prepared from control (NP) and pregnant (day 20 of pregnancy, PD20) rats. A total of 5 mg of crude nuclear extracts were incubated with radiolabelled oligonucleotides representing binding sites for HNF1-a, RXR:RAR and Stat5: (1) hepatocyte nuclear factor 1-a [(HNF1-a): gatcTGCTGGTTAATCTTTTATTT, 211/19]; (2) the retinoic acid receptor:retinoid X receptor heterodimer [(RXR:RAR):gatcTCCGGGGCATAAGGTTATGG, 256/237]; and (3) the signal transducers and activators of transcription family of transcription factors 5 [(Stat5): gatcTGTCATTCTTGGAAAAATA, 2917/2899], electrophoresed through a 6% non-denaturing polyacrylamide gel, and autoradiographed, as described in Section 2. (A) Representative electrophoretic mobility shift assays. Autoradiographs of three independent samples are shown for each group. Unlabeled specific (SP) and non-specific (NSP) competitor DNAs were included at 100-fold excess and added along with the labeled probe. Arrows show the specific bound species. The depicted autoradiographs are overnight (16 h) exposures. The experiments were repeated twice. (B) Quantitative analysis of hepatic DNA binding proteins. Gels were quantified by PhosphoImager and data (mean ^ SD) were expressed as percentage of controls. *P , 0.01, compared with controls.
study was to assess potential pregnancy-induced changes in the expression of BA transporters, we further studied the mechanisms involved in Ntcp down-regulation. Reduction of hepatic Na 1-dependent TC uptake, Ntcp protein mass, and Ntcp mRNA levels in PD20 rats appear primarily to result from a reduced rate of Ntcp gene transcription. Although we did not perform in vitro transcription studies, results from gel mobility shift assays strongly support this assumption since binding activities of two transcription factors known to be critical for Ntcp expression, HNF-1a [25] and the RXR:RAR heterodimer [12], were markedly reduced in pregnant animals. HNF-1a is a member of a family of transcription factors that are liver-
153
enriched and are involved in the regulation of a large set of genes expressed in the liver, kidney, intestine, and pancreas [26]. Recent studies using mice lacking Tcf1, the gene that codes for HNF-1a, have shown that Ntcp expression is markedly decreased (4% of that of wild-type mice) in the Tcf1- null mice underscoring the critical role of HNF-1a as a relevant transactivator of the rodent Ntcp gene [25,27]. In addition, the retinoid receptor, heterodimer RXR:RAR, has also been recently characterized as an important activator of rat Ntcp gene expression [12,28]. We have shown previously [11] that down-regulation of liver gene expression may indeed occur via reduced nuclear binding activities of critical transcriptional activators. In fact, nuclear binding activities of same two critical regulators of Ntcp promoter activity, HNF-1a and the RXR:RAR heterodimer, fall to a minimal level by 6–16 h after a single non-lethal dose of endotoxin and return to baseline levels within 48 h [11]. These changes are temporally related closely to the down-regulation of Ntcp mRNA and protein. In the present study, we found that a similar scenario, although to a lesser degree, seems to occur in the pregnant rat. Interestingly, in spite of a marked decrease of both HNF-1a and RXR:RAR heterodimer nuclear binding activities in EMSA experiments a relatively modest decrease of Ntcp mRNA and protein was found in pregnant animals. Since the binding activity of Stat5, a transcription factor which positively influences Ntcp transcription [11,29], was found to be significantly increased in pregnant rats, it is conceivable that Ntcp down-regulation during pregnancy might be attenuated to some extent by the action of this transcriptions factor. Increased activity of Stat-5 is probably related to the high levels of prolactin seen in the last third of pregnancy in mammals that is also responsible of Ntcp up-regulation during the post-partum period [29,30]. However, this assumption remains speculative. Whether Ntcp down-regulation during pregnancy is a primary or secondary phenomenon is difficult to address. Several transport processes in the liver are dramatically altered during pregnancy including hepatic uptake of organic anions [31] and reduced biliary secretion of many organic anions such as TC, dibromosulfophthalein and glucuronide conjugates of drugs [32,33]. On the other hand, Ntcp expression is decreased in the experimental model of estrogen administration [14] but there is no evidence of a direct effect of estrogen on Ntcp transcription. Recently, a wealth of information has been published with regard to how the hepatocyte maintains intracellular BA levels within a narrow concentration range and that the regulation of intracellular BA concentrations appears to be through transcriptional control of genes involved in both BA biosynthesis and transport. This is achieved through the orchestrated action of a set of several transcription factors belonging to the family of nuclear receptors in addition to RXR and RAR, namely the farsenoid X receptor (FXR), the liver X receptor, the short heterodimer partner (SHP) and others (for review see refs. [34,35]). As a
154
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
general rule, the regulatory response of hepatocytes to BA overload consists of reducing BA synthesis as well as import and increasing canalicular BA export. Thus, if canalicular excretion of BA is reduced in pregnancy, this should elicit a down-regulation of Ntcp in order to prevent further uptake of BA and protect hepatocytes from ongoing accumulation of BA. Although data from the present study and others [30] show no influence of pregnancy on the expression of the apical bile salt transporter, Bsep, functional assessment of canalicular transport of TC in the pregnant rat has shown a decreased maximal secretory rate for this bile salt [33]. Therefore, while Bsep expression is preserved in pregnant animals, its function or localization might be affected by pregnancy. This could lead to increased intracellular BA levels and Ntcp down-regulation through the activation of the BA receptor FXR, which acts as the intracellular BA sensor [35,36] and activation of SHP which in turn acts as a transcriptional repressor of Ntcp gene [37]. Although FXR activation should increase Bsep expression, it has been seen in several experimental models of cholestasis that Bsep mRNA and protein levels may not rise under conditions of intracellular BA accumulation [10], suggesting that additional mechanisms regulating Bsep expression are at play in cholestasis [9]. In fact, preliminary data from our laboratory shows that induction of Bsep gene expression by cholic acid [36] does not occur in pregnant animals (Arrese et al. unpublished results). Similar mechanisms can be postulated to occur in ethinyl-estradiol induced cholestasis where Ntcp is downregulated [9,14] and Bsep expression is relatively preserved [10] in spite of a marked decrease in ATP-dependent canalicular transport [38]. In summary, the present study shows that pregnancy is associated with a reduced expression of the major bile acid importer, Ntcp and that this phenomenon occurs at the transcriptional level, consequent to specific down-regulation of two critical regulators of Ntcp gene expression. Additionally, pregnancy is associated to a reduced expression of the main canalicular organic anion transporter Mrp2. These data provides a molecular correlate to some of the hepatobiliary changes seen during pregnancy in rodents. If these alterations are present in humans they might be related to the pathogenesis of cholestatic syndromes seen during pregnancy.
Acknowledgements This work was supported by grants from the Fondo Nacional de Ciencia y Tecnologı´a (FONDECYT #1990519 and #1020641 to MA and #1000563 to LA) and from NIH (HD20632 to FJS). Support from the Yale Liver Center is also acknowledged (DK 34989). Anti-rat Oatp-1 as well anti-rat Bsep antibodies were kindly provided by Dr Bruno Stieger (Zurich, Switzerland) and the anti-rat Mrp2 antibody was a generous gift from Dr Dietrich Keppler (Heidelberg, Germany).
References [1] Reyes H, Simon FR. Intrahepatic cholestasis of pregnancy: an estrogen-related disease. Semin Liver Dis 1993;13:289–301. [2] Vore M. Estrogen cholestasis. Membranes, metabolites, or receptors?. Gastroenterology 1987;93:643–649. [3] Vore M, Liu Y, Ganguly T. Prolactin and bile secretory function. In: Reyes HB, Leushner U, Arias IM, editors. Pregnancy, sex hormones and the liver, Dordrecht/Boston/London: Kluwer Academic Publishers, 1996. pp. 77–83. [4] Germain AM, Carvajal JA, Glasinovic JC, Kato CS, Williamson C. Intrahepatic cholestasis of pregnancy: an intriguing pregnancy-specific disorder. J Soc Gynecol Investig 2002;9:10–14. [5] Stieger B, Meier PJ. Bile salt transporters. Annu Rev Physiol 2002;64:635–661. [6] St-Pierre MV, Kullak-Ublick GA, Hagenbuch B, Meier PJ. Transport of bile acids in hepatic and non-hepatic tissues. J Exp Biol 2001;204:1673–1686. [7] Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998;273:10046– 10050. [8] Arrese M, Ananthananarayanan M, Suchy FJ. Hepatobility transport: molecular mechanisms of development and cholestasis. Pediatr Res 1998;44:141–147. [9] Lee J, Boyer JL. Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders. Semin Liver Dis 2000;20:373–384. [10] Lee JM, Trauner M, Soroka CJ, Stieger B, Meier PJ, Boyer JF. Expression of the bile salt export pump is maintained after chronic cholestasis in the rat. Gastroenterology 2000;118:163–172. [11] Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors. J Clin Invest 1998;101:2092–2100. [12] Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, Karpen SJ. Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J Biol Chem 2000;275:8835–8843. [13] Cao J, Stieger B, Meier PJ, Vore M. Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy. Am J Physiol Gastrointest Liver Physiol 2002;283:G757– G766. [14] Simon FR, Fortune J, Iwahashi M, Gartung C, Wolkoff A, Sutherland E. Ethinyl estradiol cholestasis involves alterations in expression of liver sinusoidal transporters. Am J Physiol 1996;271:G1043–G1052. [15] Icarte MA, Pizarro M, Accatino L. Adaptive regulation of hepatic bile salt transport: effects of alloxan diabetes in the rat. Hepatology 1991;14:671–678. [16] Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, et al. Down-regulation of expression and function of the rat liver Na 1/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 1996;110:199–209. [17] Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, Schlosser SF, et al. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997;113:255–264. [18] Rosario J, Sutherland E, Zaccaro L, Simon FR. Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition. Biochemistry 1988;27:3939–3946. [19] Ananthanarayanan M, Ng OC, Boyer JL, Suchy FJ. Characterization of cloned rat liver Na 1-bile acid cotransporter using peptide and fusion protein antibodies. Am J Physiol (Gastrointest Liver Physiol) 1994;267:G637–G643. [20] Chomczynski P, Sacchi N. Single-step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155 [21] Simon FR, Fortune J, Iwahashi M, Bowman S, Wolkoff A, Sutherland E. Characterization of the mechanisms involved in the gender differences in hepatic taurocholate uptake. Am J Physiol (Gastrointest Liver Physiol) 1999;276:G556–G565. [22] Reyes H, Kern Jr F. Effect of pregnancy on bile flow and biliary lipids in the hamster. Gastroenterology 1979;76:144–150. [23] Ganguly TC, Liu Y, Hyde JF, Hagenbuch B, Meier PJ, Vore M. Prolactin increases hepatic Na 1/taurocholate co-transport activity and messenger RNA post partum. Biochem J 1994;303:33–36. [24] Brites D, Rodrigues CM, van-Zeller H, Brito A, Silva R. Relevance of serum bile acid profile in the diagnosis of intrahepatic cholestasis of pregnancy in an high incidence area: Portugal. Eur J Obstet Gynecol Reprod Biol 1998;80:31–38. [25] Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, et al. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet 2001;27:375–382. [26] Pontoglio M. Hepatocyte nuclear factor 1, a transcription factor at the crossroads of glucose homeostasis. J Am Soc Nephrol 2000;11(Suppl.):S140–S143. [27] Arrese M, Karpen SJ. HNF-1 alpha: have bile acid transport genes found their ‘master’? J Hepatol 2002;36:142–145. [28] Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–147. [29] Ganguly TC, O’Brien ML, Karpen SJ, Hyde JF, Suchy FJ, Vore M. Regulation of the rat liver sodium-dependent bile acid cotransporter gene by prolactin. Mediation of transcriptional activation by Stat5. J Clin Invest 1997;15(99):2906–2914.
155
[30] Cao J, Huang L, Liu Y, Hoffman T, Stieger B, Beier PJ, et al. Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin. Hepatology 2001;33:140–147. [31] Brock WJ, Vore M. The effect of pregnancy and treatment with 17estradiol on the transport of organic anions into isolated rat hepatocytes. Drug Metab Dispos 1984;12:712–716. [32] Auansakul AC, Vore M. The effect of pregnancy and estradiol-17 beta treatment on the biliary transport maximum of dibromosulfophthalein, and the glucuronide conjugates of 5-phenyl-5-p-hydroxyphenyl[ 14C]hydantoin and [ 14C]morphine in the isolated perfused rat liver. Drug Metab Dispos 1982;10:344–349. [33] Ganguly T, Hyde JF, Vore M. Prolactin increases Na 1/taurocholate cotransport in isolated hepatocytes from postpartum rats and ovariectomized rats. J Pharmacol Exp Ther 1993;267:82–87. [34] Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 2001;30(294):1866– 1870. [35] Karpen SJ. Nuclear receptor regulation of hepatic function. J Hepatol 2002;36:832–850. [36] Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 2000;102:731–744. [37] Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–147. [38] Bossard R, Stieger B, O’Neill B, Fricker G, Meier PJ. Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver. J Clin Invest 1993;91:2714–2720.
Down-regulation of the Na 1/taurocholate cotransporting polypeptide during pregnancy in the rat Marco Arrese 1,*, Michael Trauner 2, Meenakshisundaram Ananthanarayanan 3, Margarita Pizarro 1, Nancy Solı´s 1, Luigi Accatino 1, Carol Soroka 4,5, James L. Boyer 4,5, Saul J. Karpen 6, Juan Francisco Miquel 1, Frederick J. Suchy 3 1 Department of Gastroenterology, Pontificia Universidad Cato´lica de Chile, School of Medicine, Marcoleta # 367, Santiago 6510260, Chile Division of Gastroenterology and Hepatology, Department of Internal Medicine, Karl-Franzens University School of Medicine, Graz, Austria 3 Department of Pediatrics, Laboratory of Developmental and Molecular Hepatology, Mount Sinai School of Medicine, New York, NY 10029, USA 4 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA 5 Liver Center, Yale University School of Medicine, New Haven, CT, USA 6 Texas Children’s Liver Center, Department of Pediatrics/Division of GI and Nutrition, Baylor College of Medicine, Houston, TX, USA 2
Background: Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents. Aim: To assess the expression and function of the main BA importer, the Na 1/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats. Methods: BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation. Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence. The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1-a (HNF1-a), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays. Results: A significantly reduced BA uptake and decreased Ntcp mRNA levels (240%) and protein mass (260%) was observed in pregnant rats. Nuclear extracts from pregnant rats showed a marked decrease of HNF1-a and RXR:RAR binding activities by 280 and 240% of basal activity, respectively. In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats. Conclusions: Pregnancy is associated with reduced Ntcp expression and function in the rat. Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Bile acid transport; Transporters; Na 1/taurocholate cotransporting polypeptide
1. Introduction
Received 24 April 2002; received in revised form 18 October 2002; accepted 22 October 2002 * Corresponding author. Tel.: 156-2-6863-820; fax: 156-2-6397-780. E-mail address: [email protected] (M. Arrese). Abbreviations: BA, bile acids; BSP, bromosulphthalein; Ntcp, Na 1/taurocholate cotransporting polypeptide; Oatp-1, organic anion transporting polypeptide-1; Bsep, bile salt export pump; Mrp2, multidrug resistant-associated protein; HNF1-a: hepatocyte nuclear factor 1-a; RXR:RAR, retinoid X receptor:retinoid acid receptor; Stat5, signal transducer and activator of transcription 5; EMSA, electrophoretic mobility shift assays.
Pregnancy is a physiological state known to affect bile secretory function [1]. Reduction of bile acid (BA) and bilirubin uptake as well as retention of the organic anion bromosulphthalein (BSP) have been reported in pregnant animals and similar changes have been postulated to occur in humans whose sera contain high levels of estrogens and progesterone during gestation [2,3]. These changes may have clinical implications with regard to the occurrence of clinical cholestasis during pregnancy [4].
0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S 0168 -82 78(02)00379-3
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
The recent cloning of a number of membrane transporters in hepatocytes have led to new insights into how cholephilic compounds are transported from blood to bile. Specifically, the molecular identity of transport systems involved in the transport of BA and organic anions has been elucidated [5]. Uptake of conjugated BA at the basolateral plasma membrane of the hepatocytes occurs mainly by a sodiumdependent mechanism mediated by the Na 1/taurocholate cotransporting polypeptide [Ntcp, Slc10a1]. Sodium-independent transport of non-conjugated bile acids also occurs at the basolateral membrane of hepatocytes although its physiological relevance is less clear [5]. This type of transport is thought to be mediated by members of the family of multispecific organic anion transporting polypeptides (Oatp’s) that transport unconjugated BA in a sodium-independent manner [6]. After intracellular transport, BA are excreted at the canalicular membrane of the hepatocyte mainly by an ATP-dependent mechanism that is mediated by the bile salt export pump (Bsep, Abcb11, [7]). Non-BA organic anions are mainly transported into bile by another canalicular transporter, the Multidrug resistant-associated protein 2 (Mrp2, Abcc2) that has an important function in the biliary excretion of endogenous metabolites, such as glucuronosyl-bilirubin, as well as many exogenous compounds [5,8]. Studies assessing the expression and function of hepatocyte membrane transporters during experimental conditions such as obstructive and hepatocellular cholestasis have consistently found that sinusoidal transporters such as Ntcp and Oatp-1 (Slc21a1) are downregulated during cholestatic injury [8,9]. The canalicular export pump Mrp2 is also dowregulated in various models of experimental cholestasis [9]. In contrast, changes in the canalicular BA transporter Bsep are less intense and the expression of this transporter is relatively preserved in experimental models of cholestasis including estrogen-induced cholestasis [9,10]. The present study was conducted to study the effect of pregnancy on the expression of transporters involved in hepatic BA handling by the hepatocyte in the rat in order to determine the underlying molecular mechanism of altered BA transport during this physiological state [2,3]. We also assessed the expression of the canalicular export pump Mrp2 since organic anion excretion is also affected by pregnancy in mammals [1,2]. Data presented in this communication show a significant down-regulation of the main sinusoidal BA transporter Ntcp, while Oatp-1 and Bsep expression remained unchanged. Ntcp down-regulation correlated with a significant decrease of staining in immunofluorescence studies and with a moderate decrease of BA uptake in isolated hepatocytes. Assessment of the nuclear transcription factors involved in the regulation of Ntcp [11,12] showed a significant decrease of binding activities of two critical nuclear transcription factors suggesting that pregnancy alters the transcription of Ntcp gene. Finally and in agreement with recent data [13], we found a decreased protein expression of Mrp2 in pregnant rats. Thus, down-regulation of Ntcp and
149
Mrp2 may explain some of the observed changes of bile secretory function during pregnancy in rodents 2. Material and methods 2.1. Chemicals Sodium salt of taurocholate (TC) was purchased from Sigma Chemical Company, (St. Louis, MO). [ 3H]TC (2.1 Ci/mmol) was obtained from DuPont NEN w Research Products (Boston, MA). [ 32P] deoxycytidine triphosphate was obtained from Amersham, Arlington Heights, IL. All other chemicals were of highest purity commercially available.
2.2. Animals Experiments were conducted in timed pregnant Sprague–Dawley rats weighing 210–230 g. Two time points of pregnancy were used: day 15 (PD15) and day 20 of pregnancy (PD20). The Scientific Advisory committee of the Catholic University of Chile School of Medicine approved the study protocols. Animals received humane care in compliance with the National Research Council’s criteria as outlined in Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985). Animals were anesthetized with a single dose of sodium pentobarbital (50 mg/kg body wt, intraperitoneally) and the liver removed after a short perfusion with saline solution.
2.3. Transport studies TC transport studies were performed using isolated, short-term cultured hepatocytes as described by Simon et al. [14] with minor modifications.
Fig. 1. Protein mass of hepatic bile acid transporters in male, nonpregnant female (NP) and pregnant (day 20 of pregnancy, PD20) rats. Membrane fractions were isolated from control rats and pregnant rats and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 50 mg protein/lane), and subsequently transferred to nitrocellulose membranes as described in Section 2. A representative Western blot is shown. Experiments were performed at least twice for each transporter. Molecular weights are given in kilodaltons (kDa). Densitometric analysis of protein levels showed that pregnancy is associated with a significant reduction in protein mass of Ntcp in PD20 rats while no change was seen in either Oatp-1 and Bsep protein expression (control [NP] ¼ 259 764 ^ 35 410 arbitrary densitometric units versus PD20 ¼ 77 013 ^ 9778 arbitrary densitometric units, P , 0.005). In addition, a selective decrease in Ntcp protein mass was observed in non-pregnant female rats when compared to male rats confirming previous observations on the existence of gender differences in Ntcp expression and function [21].
150
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Maximum transport of the dye BSP was performed in intact rat as previously described [15] to assess Mrp2 function.
sequence [6] and polyclonal anti-rat Mrp2 [17]. Immunoreactive bands were quantified by laser densitometry.
2.4. Tissue immunofluorescence
2.6. Messenger RNA (mRNA) studies
Qualitative distribution of Ntcp was assessed by indirect immunofluorescence as described previously [16,17]. Digital images were recorded and processed using Adobe Photoshop (Adobe Systems Inc., San Jose, CA).
Total RNA was isolated from whole-liver tissue by acid guanidinium thiocyanate-phenol chloroform extraction [20]. Poly(A)* RNA was isolated using oligo(dT)n linked to magnetic Streptavidin beads with the PolyATtract mRNA Isolation System IV (Promega, Madison, WI). Northern blots were performed using complementary DNA probes for Ntcp, Oatp-1, Bsep and Mrp2 as previously described [16,17]. The blots were exposed to Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and signals were quantitated using ImageQuant e software. Differences in loading were corrected after reprobing the stripped blots for glyceraldehyde-3phosphate dehydrogenase (GAPDH). The size of mRNA was estimated by comparison to a 0.24–9.5 Kb RNA ladder (GibcoBRL, Gaithersburg, MD).
2.5. Western analysis The protein mass of hepatic transporters in liver tissue from control and pregnant rats groups was measured as previously described [11,17] using either membrane-rich microsomal fractions, to assess the protein expression of Ntcp and Oatp-1 or canalicular-enriched liver plasma membrane fractions obtained by the method of Rosario et al. [18] to assess Bsep and Mrp2 protein mass. Additionally, Mrp2 protein expression was assessed in both plasma membrane fractions and liver homogenates as recently described by Cao et al. [13]. Western analysis was performed using standard techniques using the Renaissance Western blotting kit (New England Nuclear, Boston, MA). Membranes were probed with an anti-Ntcp fusion protein IgG as previously described [11,19], polyclonal anti-rat Oatp-1, polyclonal anti-rat Bsep antibody against a C-terminus 13-amino acid
2.7. Preparation of nuclei and nuclear protein extraction Liver nuclei were prepared from normal and PD20 animals and extracted as described previously [11]. Nuclear protein yields were similar in controls and pregnant animals.
Fig. 2. Protein mass and messenger RNA levels of Ntcp in control and pregnant rats at two time points (PD15 and PD20) during gestation: Membrane fractions were isolated from control (NP, n ¼ 3) and pregnant female rats [day 15 (PD15) and 20 (PD20) of pregnancy] and subjected to SDS-PAGE (100 mg protein/lane), and subsequently transferred to PVDF transfer membranes as described in Section 2. (A) Upper panel: Representative immunoblot. Autoradiographs of three independent samples are shown for each group. Molecular weights are given in kilo Daltons (kDa). Lower panel: Densitometric analysis of protein levels. Autoradiographs were quantified by laser densitometry, and data (mean ^ SD) are expressed as percentage of controls. *P , 0.0001 compared with controls. (B) Total mRNA was isolated as described in Section 2 and Northern blotting was performed using a radiolabelled probe for Ntcp as described in Section 2. Oatp-1 message was also quantitated for comparison. Upper panel: Representative Northern blot. Autoradiographs of three independent samples are shown for group. Each lane contains 30 mg of total RNA. Blots were stripped and reprobed for GAPDH to confirm equal loading and RNA integrity. Message sizes are given in kilobases (kb). The experiments were repeated twice. Lower panel: Quantitative analysis of Ntcp and Oatp-1 steady state RNA levels. Blots were quantified by PhosphorImager and normalized individually to GAPDH expression. Data (mean ^ SD) are expressed as Ntcp or Oatp-1/GAPDH ratio in arbitrary densitometric units. All the experiments were repeated at least twice.
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 3. Protein expression and function of the multidrug resistanceassociated protein 2 (Mrp2) in control and pregnant rats. Top: Total liver homogenate from control non-pregnant (NP) and 20 days-pregnant rats (PD20) were prepared according Cao et al. [13] and examined for the protein mass of Mrp2, the main non-bile acid organic anion canalicular transporter. Representative immunoblot showing a 50% decrease in protein mass of Mrp2 in pregnant rats compared with controls (NP: 628 353 ^ 12 099 arbitrary densitometric units in NP versus 315 444 ^ 4348 in PD20, n ¼ 3 in each group, P , 0.02). Bottom: Hepatobiliary transport of the model Mrp2 substrate BSP in NP and PD20 rats. BSP was infused through the jugular vein in a stepwiseincreasing rates as described [15]. Maximum secretory rate was calculated as the mean of the three highest consecutive values of BSP secretion and expressed as micrograms of BSP per minute per gram of liver. A significant decrease in BSP was observed in pregnant rats compared with controls (29.4 ^ 4.6 versus 19.2 ^ 1.4, in NP (n ¼ 5) and PD20 (n ¼ 6) rats, respectively, P , 0.05).
2.8. Electrophoretic gel mobility shift assays A total of 2–10 mg of nuclear extracts were incubated on ice for 30 min with 2 £ 10 4 cpm of 32P end-labeled oligonucleotide as described previously [11]. Specific oligonucleotides (sense strands sequences with their position in rat ntcp promoter are shown in Fig. 5) were used in electrophoretic mobility shift assays (EMSA) to assess the binding activities of transcription factors. Double-stranded oligonucleotide probes were endlabeled and purified according to standard procedures. In competition assays, 100-fold molar excess of the specific unlabeled oligonucleotide was added to the binding mixtures along with the labeled oligonucleotide.
151
tic transporters at the protein level, immunoblotting was performed in microsomal-enriched (analysis of Ntcp and Oatp-1) and canalicular membrane fractions (analysis of Bsep and Mrp2). Representative Western blots are shown in Figs. 1–3. Pregnancy was associated with a significant reduction in Ntcp protein mass in PD15 and PD20 rats while no change was seen in either Oatp-1 or Bsep protein expression. In addition, a selective decrease in Ntcp protein mass was also observed in non-pregnant females when compared to male rats. This confirms previous observations showing that Ntcp is dimorphically expressed in rat liver due to gender differences in transcription with males exhibiting greater amounts of Ntcp [21]. Analysis of Mrp2 protein expression showed a decrease in the protein mass of this transporter in agreement with data from Cao et al. [13]. Although we consistently reproduced this result using both liver homogenates and plasma membrane fractions, best quality Western blots were obtained with liver homogenates prepared following the methods reported by Cao et al. [13]. Therefore, representative blots of Mrp2 are shown in Fig. 3. 3.2. Tissue immunofluorescence Qualitative distribution of hepatic transporters was studied by indirect immunofluorescence in control and pregnant rats (PD20). Fig. 4 shows representative images of immunostaining with Ntcp antibody demonstrating a decreased labeling of the sinusoidal membrane in livers from PD20 rats. In addition, and in agreement with data from Cao et al. [13], a decreased immunostaining for Mrp2 was also found in pregnant rats. 3.3. mRNA studies Steady state mRNA levels for Ntcp, Oatp-1, Bsep and Mrp2 were assessed by Northern blot analysis. Densitometric analysis using the Storm 860 Phosphorimager apparatus showed a significant reduction of liver Ntcp mRNA levels by approximately 40% in both PD15 and PD20 rats, when compared to non-pregnant controls (Fig. 2B). No changes were seen in steady state mRNA levels for Oatp1, one of the Na 1-independent basolateral organic anion transporters or the canalicular ATP-dependent transporters Bsep and Mrp2 (data not shown) at both time points studied.
2.9. Statistics 3.4. Transport studies All results are expressed as mean ^ SE. A two-tailed non-paired Student’s t-test was used to compare differences between groups. Values were considered significantly different when the P value was equal to or less than 0.05.
3. Results 3.1. Western analysis To determine if pregnancy affects the expression of hepa-
In order to investigate if pregnancy is associated with a reduced function of Ntcp, [ 3H]TC uptake was assessed in short-term cultured hepatocytes from control and PD20 rats. Sodium-dependent [ 3H]TC uptake was reduced by 38% in pregnant animals as compared to non-pregnant rats (322.66 ^ 60 pmol mg protein 1 30 s 21 in controls versus 200.04 ^ 15 pmol mg protein 21 30 s 21 in PD20 rats, mean ^ SE n ¼ 3 in each group, t-statistic 3.4, P , 0:05) while sodium independent uptake was essentially
152
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 4. Indirect immunofluorescent localization of Ntcp and Mrp2 in control and pregnant rats. Frozen liver sections from control non-pregnant (NP) and 20 days-pregnant rats (PD20) were used to assess qualitative distribution of Ntcp and Mrp2 by indirect immunofluorescence as described previously [16,17]. A decreased labeling of both sinusoidal and canalicular membrane was observed accounting for reduction in Ntcp and Mrp2 protein expression in pregnant rats. Superimposition of images is shown on the right.
unchanged (71.3 ^ 8.3 pmol mg protein 1 30 s 1 in controls versus 75.37 ^ 15.6 pmol mg protein 1 30 s 1 in PD20 rats, mean ^ SE n ¼ 3 in each group, P . 0:05). In addition, decreased expression of Mrp2 during pregnancy was associated to a reduced maximum transport of the dye BSP in pregnant rats (Fig. 3).
the three oligonucleotide probes, the specificity of the DNAprotein interactions was demonstrated by appropriate competition assays (Fig. 5).
3.5. Binding activities of critical transcription factors of Ntcp promoter
Ntcp is the major bile acid uptake system in the basolateral membrane of rat hepatocytes [5]. The expression of Ntcp gene is significantly down-regulated in obstructive as well as hepatocellular cholestasis (including estrogeninduced) in rats [9,14,16]. The aim of this study was to determine if the expression of the major BA transport proteins were altered during pregnancy, since reduced BA transport has been reported during this physiological state in rodents [22,23]. A significant down-regulation of the protein and mRNA levels of the principal BA importer, Ntcp was seen during late pregnancy, while no changes were seen in the expression of another sinusoidal transporter, Oatp-1 nor the canalicular BA export pump Bsep. Our data suggest that the decrease of Ntcp is related to a reduced activity of two critical transcriptional regulators of the Ntcp promoter (see Fig. 5). Ntcp down-regulation during pregnancy provides a molecular mechanism for the reduced BA uptake occurring in the pregnant rat [23] corresponding to increased levels of serum BA seen during pregnancy in humans [24]. In addition to Ntcp down-regulation, we also found a decrease in the protein expression and function of the main canalicular organic bile acid transporter Mrp2, in agreement with recently published data [13]. Since the original aim of this
Multiple elements in the rat Ntcp promoter are bound by positive-acting transcription factors: an hepatocyte nuclear factor 1-a (HNF1-a) site at 27/18, a retinoic acid-response element (250/237), and tandem signal transducer and activator of transcription 5 (Stat5) sites (2936/2928 and 2912/ 2904). EMSA were performed using nuclear extracts from PD20 rats in order to address whether Ntcp down-regulation observed during pregnancy was related to decreased binding activities of some of these transactivators of transcription in the Ntcp promoter as previously reported by us in the endotoxin-induced cholestasis model [9,11]. Fig. 5 shows results of these experiments. A marked decrease of HNF1-a and retinoid X receptor:retinoid acid receptor (RXR:RAR) binding activities was found (277 and 260% compared to basal levels respectively, P , 0:01). In contrast, binding activity of Stat-5 was significantly increased by 150% in nuclear extracts from pregnant rats. These findings indicate that nuclei prepared from pregnant rats contain selectively decreased amounts of active HNF1-a and RXR:RAR levels and increased amounts of Stat5 transcription factor. For all
4. Discussion
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
Fig. 5. Reduced binding activities of transcriptional regulators of Ntcp promoter during pregnancy. Hepatic nuclear extracts were prepared from control (NP) and pregnant (day 20 of pregnancy, PD20) rats. A total of 5 mg of crude nuclear extracts were incubated with radiolabelled oligonucleotides representing binding sites for HNF1-a, RXR:RAR and Stat5: (1) hepatocyte nuclear factor 1-a [(HNF1-a): gatcTGCTGGTTAATCTTTTATTT, 211/19]; (2) the retinoic acid receptor:retinoid X receptor heterodimer [(RXR:RAR):gatcTCCGGGGCATAAGGTTATGG, 256/237]; and (3) the signal transducers and activators of transcription family of transcription factors 5 [(Stat5): gatcTGTCATTCTTGGAAAAATA, 2917/2899], electrophoresed through a 6% non-denaturing polyacrylamide gel, and autoradiographed, as described in Section 2. (A) Representative electrophoretic mobility shift assays. Autoradiographs of three independent samples are shown for each group. Unlabeled specific (SP) and non-specific (NSP) competitor DNAs were included at 100-fold excess and added along with the labeled probe. Arrows show the specific bound species. The depicted autoradiographs are overnight (16 h) exposures. The experiments were repeated twice. (B) Quantitative analysis of hepatic DNA binding proteins. Gels were quantified by PhosphoImager and data (mean ^ SD) were expressed as percentage of controls. *P , 0.01, compared with controls.
study was to assess potential pregnancy-induced changes in the expression of BA transporters, we further studied the mechanisms involved in Ntcp down-regulation. Reduction of hepatic Na 1-dependent TC uptake, Ntcp protein mass, and Ntcp mRNA levels in PD20 rats appear primarily to result from a reduced rate of Ntcp gene transcription. Although we did not perform in vitro transcription studies, results from gel mobility shift assays strongly support this assumption since binding activities of two transcription factors known to be critical for Ntcp expression, HNF-1a [25] and the RXR:RAR heterodimer [12], were markedly reduced in pregnant animals. HNF-1a is a member of a family of transcription factors that are liver-
153
enriched and are involved in the regulation of a large set of genes expressed in the liver, kidney, intestine, and pancreas [26]. Recent studies using mice lacking Tcf1, the gene that codes for HNF-1a, have shown that Ntcp expression is markedly decreased (4% of that of wild-type mice) in the Tcf1- null mice underscoring the critical role of HNF-1a as a relevant transactivator of the rodent Ntcp gene [25,27]. In addition, the retinoid receptor, heterodimer RXR:RAR, has also been recently characterized as an important activator of rat Ntcp gene expression [12,28]. We have shown previously [11] that down-regulation of liver gene expression may indeed occur via reduced nuclear binding activities of critical transcriptional activators. In fact, nuclear binding activities of same two critical regulators of Ntcp promoter activity, HNF-1a and the RXR:RAR heterodimer, fall to a minimal level by 6–16 h after a single non-lethal dose of endotoxin and return to baseline levels within 48 h [11]. These changes are temporally related closely to the down-regulation of Ntcp mRNA and protein. In the present study, we found that a similar scenario, although to a lesser degree, seems to occur in the pregnant rat. Interestingly, in spite of a marked decrease of both HNF-1a and RXR:RAR heterodimer nuclear binding activities in EMSA experiments a relatively modest decrease of Ntcp mRNA and protein was found in pregnant animals. Since the binding activity of Stat5, a transcription factor which positively influences Ntcp transcription [11,29], was found to be significantly increased in pregnant rats, it is conceivable that Ntcp down-regulation during pregnancy might be attenuated to some extent by the action of this transcriptions factor. Increased activity of Stat-5 is probably related to the high levels of prolactin seen in the last third of pregnancy in mammals that is also responsible of Ntcp up-regulation during the post-partum period [29,30]. However, this assumption remains speculative. Whether Ntcp down-regulation during pregnancy is a primary or secondary phenomenon is difficult to address. Several transport processes in the liver are dramatically altered during pregnancy including hepatic uptake of organic anions [31] and reduced biliary secretion of many organic anions such as TC, dibromosulfophthalein and glucuronide conjugates of drugs [32,33]. On the other hand, Ntcp expression is decreased in the experimental model of estrogen administration [14] but there is no evidence of a direct effect of estrogen on Ntcp transcription. Recently, a wealth of information has been published with regard to how the hepatocyte maintains intracellular BA levels within a narrow concentration range and that the regulation of intracellular BA concentrations appears to be through transcriptional control of genes involved in both BA biosynthesis and transport. This is achieved through the orchestrated action of a set of several transcription factors belonging to the family of nuclear receptors in addition to RXR and RAR, namely the farsenoid X receptor (FXR), the liver X receptor, the short heterodimer partner (SHP) and others (for review see refs. [34,35]). As a
154
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155
general rule, the regulatory response of hepatocytes to BA overload consists of reducing BA synthesis as well as import and increasing canalicular BA export. Thus, if canalicular excretion of BA is reduced in pregnancy, this should elicit a down-regulation of Ntcp in order to prevent further uptake of BA and protect hepatocytes from ongoing accumulation of BA. Although data from the present study and others [30] show no influence of pregnancy on the expression of the apical bile salt transporter, Bsep, functional assessment of canalicular transport of TC in the pregnant rat has shown a decreased maximal secretory rate for this bile salt [33]. Therefore, while Bsep expression is preserved in pregnant animals, its function or localization might be affected by pregnancy. This could lead to increased intracellular BA levels and Ntcp down-regulation through the activation of the BA receptor FXR, which acts as the intracellular BA sensor [35,36] and activation of SHP which in turn acts as a transcriptional repressor of Ntcp gene [37]. Although FXR activation should increase Bsep expression, it has been seen in several experimental models of cholestasis that Bsep mRNA and protein levels may not rise under conditions of intracellular BA accumulation [10], suggesting that additional mechanisms regulating Bsep expression are at play in cholestasis [9]. In fact, preliminary data from our laboratory shows that induction of Bsep gene expression by cholic acid [36] does not occur in pregnant animals (Arrese et al. unpublished results). Similar mechanisms can be postulated to occur in ethinyl-estradiol induced cholestasis where Ntcp is downregulated [9,14] and Bsep expression is relatively preserved [10] in spite of a marked decrease in ATP-dependent canalicular transport [38]. In summary, the present study shows that pregnancy is associated with a reduced expression of the major bile acid importer, Ntcp and that this phenomenon occurs at the transcriptional level, consequent to specific down-regulation of two critical regulators of Ntcp gene expression. Additionally, pregnancy is associated to a reduced expression of the main canalicular organic anion transporter Mrp2. These data provides a molecular correlate to some of the hepatobiliary changes seen during pregnancy in rodents. If these alterations are present in humans they might be related to the pathogenesis of cholestatic syndromes seen during pregnancy.
Acknowledgements This work was supported by grants from the Fondo Nacional de Ciencia y Tecnologı´a (FONDECYT #1990519 and #1020641 to MA and #1000563 to LA) and from NIH (HD20632 to FJS). Support from the Yale Liver Center is also acknowledged (DK 34989). Anti-rat Oatp-1 as well anti-rat Bsep antibodies were kindly provided by Dr Bruno Stieger (Zurich, Switzerland) and the anti-rat Mrp2 antibody was a generous gift from Dr Dietrich Keppler (Heidelberg, Germany).
References [1] Reyes H, Simon FR. Intrahepatic cholestasis of pregnancy: an estrogen-related disease. Semin Liver Dis 1993;13:289–301. [2] Vore M. Estrogen cholestasis. Membranes, metabolites, or receptors?. Gastroenterology 1987;93:643–649. [3] Vore M, Liu Y, Ganguly T. Prolactin and bile secretory function. In: Reyes HB, Leushner U, Arias IM, editors. Pregnancy, sex hormones and the liver, Dordrecht/Boston/London: Kluwer Academic Publishers, 1996. pp. 77–83. [4] Germain AM, Carvajal JA, Glasinovic JC, Kato CS, Williamson C. Intrahepatic cholestasis of pregnancy: an intriguing pregnancy-specific disorder. J Soc Gynecol Investig 2002;9:10–14. [5] Stieger B, Meier PJ. Bile salt transporters. Annu Rev Physiol 2002;64:635–661. [6] St-Pierre MV, Kullak-Ublick GA, Hagenbuch B, Meier PJ. Transport of bile acids in hepatic and non-hepatic tissues. J Exp Biol 2001;204:1673–1686. [7] Gerloff T, Stieger B, Hagenbuch B, Madon J, Landmann L, Roth J, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998;273:10046– 10050. [8] Arrese M, Ananthananarayanan M, Suchy FJ. Hepatobility transport: molecular mechanisms of development and cholestasis. Pediatr Res 1998;44:141–147. [9] Lee J, Boyer JL. Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders. Semin Liver Dis 2000;20:373–384. [10] Lee JM, Trauner M, Soroka CJ, Stieger B, Meier PJ, Boyer JF. Expression of the bile salt export pump is maintained after chronic cholestasis in the rat. Gastroenterology 2000;118:163–172. [11] Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors. J Clin Invest 1998;101:2092–2100. [12] Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, Karpen SJ. Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J Biol Chem 2000;275:8835–8843. [13] Cao J, Stieger B, Meier PJ, Vore M. Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy. Am J Physiol Gastrointest Liver Physiol 2002;283:G757– G766. [14] Simon FR, Fortune J, Iwahashi M, Gartung C, Wolkoff A, Sutherland E. Ethinyl estradiol cholestasis involves alterations in expression of liver sinusoidal transporters. Am J Physiol 1996;271:G1043–G1052. [15] Icarte MA, Pizarro M, Accatino L. Adaptive regulation of hepatic bile salt transport: effects of alloxan diabetes in the rat. Hepatology 1991;14:671–678. [16] Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, et al. Down-regulation of expression and function of the rat liver Na 1/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 1996;110:199–209. [17] Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, Schlosser SF, et al. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997;113:255–264. [18] Rosario J, Sutherland E, Zaccaro L, Simon FR. Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition. Biochemistry 1988;27:3939–3946. [19] Ananthanarayanan M, Ng OC, Boyer JL, Suchy FJ. Characterization of cloned rat liver Na 1-bile acid cotransporter using peptide and fusion protein antibodies. Am J Physiol (Gastrointest Liver Physiol) 1994;267:G637–G643. [20] Chomczynski P, Sacchi N. Single-step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–159.
M. Arrese et al. / Journal of Hepatology 38 (2003) 148–155 [21] Simon FR, Fortune J, Iwahashi M, Bowman S, Wolkoff A, Sutherland E. Characterization of the mechanisms involved in the gender differences in hepatic taurocholate uptake. Am J Physiol (Gastrointest Liver Physiol) 1999;276:G556–G565. [22] Reyes H, Kern Jr F. Effect of pregnancy on bile flow and biliary lipids in the hamster. Gastroenterology 1979;76:144–150. [23] Ganguly TC, Liu Y, Hyde JF, Hagenbuch B, Meier PJ, Vore M. Prolactin increases hepatic Na 1/taurocholate co-transport activity and messenger RNA post partum. Biochem J 1994;303:33–36. [24] Brites D, Rodrigues CM, van-Zeller H, Brito A, Silva R. Relevance of serum bile acid profile in the diagnosis of intrahepatic cholestasis of pregnancy in an high incidence area: Portugal. Eur J Obstet Gynecol Reprod Biol 1998;80:31–38. [25] Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, et al. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet 2001;27:375–382. [26] Pontoglio M. Hepatocyte nuclear factor 1, a transcription factor at the crossroads of glucose homeostasis. J Am Soc Nephrol 2000;11(Suppl.):S140–S143. [27] Arrese M, Karpen SJ. HNF-1 alpha: have bile acid transport genes found their ‘master’? J Hepatol 2002;36:142–145. [28] Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–147. [29] Ganguly TC, O’Brien ML, Karpen SJ, Hyde JF, Suchy FJ, Vore M. Regulation of the rat liver sodium-dependent bile acid cotransporter gene by prolactin. Mediation of transcriptional activation by Stat5. J Clin Invest 1997;15(99):2906–2914.
155
[30] Cao J, Huang L, Liu Y, Hoffman T, Stieger B, Beier PJ, et al. Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin. Hepatology 2001;33:140–147. [31] Brock WJ, Vore M. The effect of pregnancy and treatment with 17estradiol on the transport of organic anions into isolated rat hepatocytes. Drug Metab Dispos 1984;12:712–716. [32] Auansakul AC, Vore M. The effect of pregnancy and estradiol-17 beta treatment on the biliary transport maximum of dibromosulfophthalein, and the glucuronide conjugates of 5-phenyl-5-p-hydroxyphenyl[ 14C]hydantoin and [ 14C]morphine in the isolated perfused rat liver. Drug Metab Dispos 1982;10:344–349. [33] Ganguly T, Hyde JF, Vore M. Prolactin increases Na 1/taurocholate cotransport in isolated hepatocytes from postpartum rats and ovariectomized rats. J Pharmacol Exp Ther 1993;267:82–87. [34] Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 2001;30(294):1866– 1870. [35] Karpen SJ. Nuclear receptor regulation of hepatic function. J Hepatol 2002;36:832–850. [36] Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 2000;102:731–744. [37] Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001;121:140–147. [38] Bossard R, Stieger B, O’Neill B, Fricker G, Meier PJ. Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver. J Clin Invest 1993;91:2714–2720.