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Endocrinology Vol. 142, No. 10 4212-4222
Copyright © 2001 by The Endocrine Society

ARTICLES

PRL, Placental Lactogen, and GH Induce Na+/Taurocholate-Cotransporting Polypeptide Gene Expression by Activating Signal Transducer and Activator of Transcription-5 in Liver Cells

Jingsong Cao, P. Mangala Gowri, Tanmoy C. Ganguly, Marcie Wood, James F. Hyde, Frank Talamantes and Mary Vore

Graduate Center for Toxicology (J.C., P.M.G., T.C.G., M.W., M.V.) and Department of Anatomy and Neurobiology (J.F.H.), College of Medicine, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0305; and Department of Biology (F.T.), University of California, Santa Cruz, California 95064

Address all correspondence and requests for reprints to: Mary Vore, Graduate Center for Toxicology, 306 Health Science Research Building, University of Kentucky, Lexington, Kentucky 40536-0305. E-mail: maryv{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the transcriptional regulation of the Na+/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na+/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na+/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na+/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na+/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na+/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL, GH, AND placental lactogens (PLs) constitute a family of hormones believed to have arisen from a common ancestral gene by two successive tandem duplications (1). PRL and GH are secreted by the anterior pituitary; whereas in rodents, PLs are produced by trophoblast giant cells (2, 3). PRL is usually involved in reproduction, lactation, water-salt balance, growth and development, behavior, and the immune response (4); whereas GH plays a central role in regulating somatic growth and intermediary metabolism in vertebrates (5). Depending on the species, PLs exert GH- or PRL-like effects in both maternal and fetal tissues (1). The biological activities of PRL, GH, and PL are mediated by specific membrane receptors (1). In general, GH mediates its actions by binding to the GH receptor (GHR), whereas the PRL receptor (PRLR) is specific for PRL and PL (1, 4, 6).

The similar signal transduction mechanisms involved in PRL and GH actions have been extensively studied (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Binding of each hormone to its cognate receptor leads to receptor dimerization and activation of the intracellular tyrosine kinase, Janus kinase 2 (Jak2) (10, 17, 18, 19). The activated Jak2, in turn, phosphorylates the receptor at specific tyrosine residues within the cytoplasmic domain, thus recruiting members of the signal transducers and activators of transcription (Stat) protein family, including Stat1, -3, and -5 (5, 16, 20). Jak-mediated tyrosine phosphorylation of the receptor-bound Stats leads to their dissociation from the receptor, dimerization by means of reciprocal SH2-phosphotyrosine interactions, and translocation to the nucleus, where the Stat dimers bind to specific DNA sequences and induce gene transcription. A similar Jak2-Stat5 signal transduction pathway for mouse PL (mPL)-I via PRLR was also demonstrated in Nb2 cells (21).

In the rat, there are two different forms of PRLR, which differ in the length of the cytoplasmic domain. The long form (591aa) (PRLRL) predominates in the mammary gland and ovary, whereas the short form (291aa) (PRLRS) is the major form expressed in liver (22). PRLRS lacks the cytoplasmic domain and is not able to transduce the PRL signal via Stat5 (9). Studies have shown that the PRLRS may serve as a dominant negative isoform and prevent signal transduction via the PRLRL (23). However, the levels and ratio of PRLRS to PRLRL vary in the female rat liver and are modulated by physiological conditions such as pregnancy and lactation as well as by PRL treatment (24, 25). These data, coupled with the high concentration of PRLR in female rat liver, have suggested a role for PRL in regulating liver function.

A critical function of the mammalian liver is the formation and maintenance of bile flow. The active vectorial transport of osmotically active solutes (primarily bile salts) and accompanying counterions from plasma to the bile canaliculus, followed by the passive movement of water until osmotic equilibrium is reached, is the basis for the formation of bile flow (26). Bile serves as an important route for excretion of cholesterol, metabolites of drugs, and endogenous waste products such as bilirubin and steroid metabolites and of bile salts, which are essential for the emulsification and absorption of fats and fat-soluble vitamins from the intestinal tract. Bile salts are synthesized exclusively in the liver and are transported across the canalicular membrane of the hepatocytes to bile by a primary active, ATP-dependent transporter termed the bile salt export pump (bsep) (27). Bile salts are absorbed throughout the small intestine, with the principal component being an efficient, active, sodium-dependent transporter residing on the apical surface of ileal enterocytes (28). Bile salts are then returned to the liver via the portal circulation, and are taken up across the basolateral domain of the hepatocytes, primarily (85%) by the Na+/taurocholate (TC) cotransporting polypeptide (ntcp) (29). PRL plays a critical role in increasing maternal bile secretory function postpartum (30) and increases Na+/TC cotransport activity that is caused by increases in ntcp mRNA and protein expression (31, 32). The expression of bsep is also increased postpartum as a result of the actions of PRL (33). By using a postpartum suckling rat model, we showed that the suckling stimulus, which induces a striking elevation in plasma PRL levels, leads to nuclear translocation of the phosphorylated Stat5 in the liver, which binds to two Stat5 response elements in the promoter of the ntcp gene. Cotransfection studies in HepG2 cells demonstrated that PRL acts via PRLRL to activate Stat5 and increase transcription of ntcp (34). However, suckling can also stimulate the release of GH, albeit in much smaller amounts (35), which activates Stat5 via the GHR (16). This raised the question as to whether GH or PRL, or both, were responsible for the activation of liver Stat5 seen in suckled female rats. Administration of GH, but not PRL, to adult (nonsuckled) females leads to activation of Stat5a and Stat5b in the liver (36). In ovariohysterectomized pregnant rats administered PRL (250 µg, ip), EMSAs detected increased activated Stat5 in mammary gland; whereas in liver, activated Stat5 was detected in control animals but was only slightly modified by PRL treatment (37). These findings have led to the impression that the liver lacks some critical component essential for PRL-mediated activation of the Jak2/Stat5 pathway, and stimulated us to investigate further whether PRL could activate the Jak2/Stat5 pathway in hepatocytes and thus increase ntcp expression.

In rodents, PRL levels decline in pregnancy (4, 38), whereas estradiol levels are elevated (39). High doses of estrogens suppress ntcp expression and activity (40) and might thus be expected to reduce ntcp expression during pregnancy. However, both ntcp mRNA and protein levels are maintained in pregnancy with little (~10%) or no suppression (31, 33). The high plasma levels of PLs during the latter half of pregnancy (38) and the specific binding of PL-I to maternal liver suggest a role for the PLs in the regulation of maternal intermediary metabolism (41), leading us to postulate that the PLs play a role in maintaining the expression of ntcp in pregnancy.

In this study, we used freshly isolated rat hepatocytes to demonstrate transactivation of the ntcp gene via the Stat5 pathway by PRL, GH, and PL-I. We also investigated the ability of GH and PL-I to activate the ntcp gene, using the HepG2 cell transient cotransfection assay, relative to PRL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ovine PRL (NIDDK-oPRL-21; AFP10692C) and rat GH (rGH) were provided by the NIDDK, the National Hormone and Pituitary Program, and Dr. A. F. Parlow (National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA). Recombinant mPL-I was prepared as described (42). Antibodies used in this study were polyclonal rabbit antimouse antibodies to Stat5, Stat1, Stat3, phosphotyrosine agarose conjugated (PY-20AC) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal rabbit antimouse antibodies to Stat5a, Stat5b (Zymed Laboratories, Inc., South San Francisco, CA), and donkey antirabbit IgG horseradish peroxidase conjugates (Amersham Pharmacia Biotech, Arlington Heights, IL). The expression vector containing the rat GHR (rGHR) cDNA, pcDNA-rGHR, and ovine Stat5a cDNA, pXM-Stat5a, were kindly provided by Dr. B. Groner (Institute for Experimental Cancer Research, Freiburg, Germany) (43). The cDNA for the mouse GHR (mGHR) was a generous gift from Dr. John Kopchick (Edison Biotechnology Institute Ohio University, Athens, OH) and was cloned into the XhoI/XbaI site of the vectors pcDNA 3.1 (-) (Invitrogen, Carlsbad, CA). The cDNAs for rPRLRL and rPRLRS were a gift from Dr. Paul Kelly (INSERM, Paris, France) and were prepared as described (34). Plasmid pRSV-ß-galactosidase was kindly provided by Dr. Daniel Noonan of the University of Kentucky. The ntcp luciferase reporter construct 4 x 0.2pGL3 with the ntcp minimal promoter (-158 to + 47) linked to four Stat5 response elements (TTCTTGGAA) and the native ntcp promoter construct p1237Luc were prepared as reported earlier (34). Plasmid 0.5pT109Luc was constructed by inserting the 500-bp HindIII fragment of the ntcp promoter (-1237 to -758, containing the two native Stat5 response elements) upstream to the herpes simplex virus thymidine kinase promoter in pT109Luc (American Type Culture Collection, Manassas, VA). Plasmid DNA was extracted using the QIAGEN (Chatsworth, CA) midicolumns, or by CsCl gradient centrifugation twice. Polynucleotide kinase was obtained from Life Technologies, Inc. (Gaithersburg, MD). Genistein was purchased from Sigma (St. Louis, MO).

Animals
Female Sprague Dawley rats (200–250 g) (Harlan Sprague Dawley, Inc., Indianapolis, IN) were used throughout. Pregnant rats were timed according to the first day that sperm was detected (d 0). Rats at 19–20 d of pregnancy were used as pregnant rats. The rats had free access to food and water and were maintained on an automatically timed 12-h light, 12-h dark cycle. All protocols dealing with animals were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and followed the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Transient transfections
HepG2 cells were maintained in DMEM/F12 (1:1) medium, supplemented with 10% FBS (Life Technologies, Inc.), 3.58 mM glutamine, 55 µg/ml gentamicin, and 1 µg/ml insulin (Life Technologies, Inc.). A day before transfection, the cells were subcultured into phenol-red-free DMEM supplemented with 10% charcoal-stripped FBS (HyClone Laboratories, Inc., Logan, UT), 3.58 mM glutamine, and 55 µg/ml gentamicin. Transfections were carried out with CsCl or column-purified DNA. Cells were transfected with 5 µg ntcp luciferase construct 4 x 0.2pGL3, p1237Luc, or 0.5pT109Luc, 5 µg pXM-Stat5a, and 1 µg indicated receptors cDNA [pL3-PRLRL or PL3-PRLRS or pcDNA-rGHR or pcDNA3.1(-)-mGHR]. pRSV-ßgal (5 µg) was included as a control, with pUC19 as a carrier, to a total of 20 µg/10-cm plate. In rPRLRL and rGHR cotransfection experiments, 1 µg pL3-PRLRL and 1 µg or 4 µg pcDNA-rGHR were used. The calcium phosphate-DNA coprecipitation method was followed for transfections (44). Six to 8 h after transfection, the medium was removed and the cells were washed twice with PBS and replated in a 96-well plate. The cells were treated with blank medium or the indicated concentrations of ligands (mPL-I, oPRL, rGH, hGH); and, after a further incubation of 36–44 h, the cells washed with PBS and lysed with 50 µl lysis buffer for 20 min at room temperature. The cell extract (20 µl) was combined with 100 µl K-ATP/MgCl2 buffer and assayed for luciferase activity (Microlumat LB 96P, EG&G Berthold, Bad Wildbad, Germany). The remaining cell extract was mixed with 200 µl o-nitrophenyl ß-galactopyranoside substrate solution and analyzed for ß-galactosidase activity at 415 nm on an ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, CA). The composition of all the solutions and buffers used in the assays was as reported earlier (34). The normalized luciferase response was calculated as relative light units/ß-galactosidase activity (A at 415 nm)·min. Single transfections were conducted at least in duplicate, and the mean was calculated for each data point. The hormone-dependent fold-induction (relative to the no-hormone exposure as control) is represented as mean ± SEM for three to five independent transfections.

Isolation and culture of rat hepatocytes
Hepatocytes were isolated by a two-step perfusion method as described previously (45). The hepatocyte suspension was added to 60-mm dishes precoated with rat tail collagen I matrix (12.5 µg/cm2) at a density of 2 x 106 cells/dish and incubated in a humidified atmosphere of 5% CO2 in air at 37 C. Dishes were precoated with collagen diluted in HBSS (125 µg/ml collagen I; 3 ml/dish), incubated overnight at 37 C, and washed with Williams E (WE) medium before use. After 2–3 h incubation, the unattached cells were poured off by washing once with 5 ml WE medium containing 5% FBS. The attached cells were exposed to various concentrations of hormones in 5 ml WE medium containing 5% FBS or blank medium for an additional 0.5–120 min. In some experiments, cells were pretreated with the tyrosine kinase inhibitor genistein (20, 50, or 100 µM) for 1 h before exposure to oPRL.

Preparation of nuclear extracts
After the termination of treatments, HepG2 cells or hepatocytes were washed twice with PBS and scraped into 1.5 ml PBS and centrifuged at 2,000 x g for 30 sec. The resulting cell pellet was resuspended in a hypotonic buffer [10 mM HEPES, pH 7.9;1.5 mM MgCl2; 10 mM KCl; 1 mM Na3VO4; 1 mM NaF; 0.5 mM dithiothreitol; 0.2 mM phenylmethylsulfonylfluoride (PMSF); 1 µg/ml pepstatin A; 5 µg/ml aprotinin; 2 µg/ml leupeptin; and 5 µg/ml antipain]. The suspension was incubated by gently rotating at 4 C for 30 min and then was centrifuged at 5,000 x g for 1 min. The pellet was resuspended in a hypertonic buffer (20 mM HEPES, pH 7.9; 25% glycerol; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA, pH 8.0; 1 mM Na3VO4; 1 mM NaF; 0.5 mM dithiothreitol; 0.2 mM PMSF; and the protease inhibitors as described above) and rotated end-over-end at 4 C for 60 min. The extract was centrifuged at 10,000 x g for 30 min, and aliquots of the supernatant were stored at -70 C for further use. Protein concentrations were measured by the method of Lowry (46) using BSA as standard.

Immunoprecipitation and Western blots
For immunoprecipitation, 75 µg nuclear protein was incubated with 10 µg mouse monoclonal antiphosphotyrosine agarose conjugate in 200 µl immunoprecipitation buffer (20 mM HEPES, pH 7.9; 1% Nonidet P-40; 10% glycerol; 2.5 mM EDTA, pH 8.0; 2.5 mM EGTA; 0.5 mM Na3VO4; 0.5 mM NaF; 1 mM PMSF; 2 µg/ml pepstatin A; 2 µg/ml aprotinin; 10 µg/ml leupeptin; 50 µg/ml antipain; 2 µg/ml chymostatin; and 10 µg/ml trypsin inhibitor) at 4 C overnight by rotating end over end. The protein agarose conjugates were pelleted and washed three times with immunoprecipitation buffer. The protein was eluted from the beads by boiling for 5 min in 40 µl 2 x SDS loading buffer (125 mM Tris/HCl, pH 6.8; 2.5% SDS; 20% glycerol; 5% ß-mercaptoethanol; and 0.005% bromophenol blue). For Western blots, 10 µl of the above immunoprecipitated proteins were resolved on an 8.5% SDS-PAGE and then transferred to nitrocellulose membrane. The membrane was incubated at 4 C overnight in washing buffer (0.9% NaCl; 20 mM Tris/HCl, pH 7.5; 0.1% Tween-20) containing 5% nonfat milk to block nonspecific binding. The blots were then exposed to primary antibodies (0.5 µg/ml anti-Stats) dissolved in the same buffer for 1 h at room temperature. After 4 washes (5 min each), the membranes were incubated with antirabbit IgG horseradish peroxidase conjugate (1:4000) for 1 h at room temperature. After another four 5-min washes, the blots were visualized using ECL+Plus (Amersham Pharmacia Biotech) for 5 min and exposed to x-ray film for 1–30 min.

EMSAs
The EMSAs were conducted as described (34). Three double-stranded oligonucleotides were used (sense strand sequence; protein-binding sites are italicized): a 21 oligomer containing the bovine ß-casein Stat5 consensus binding motif (5'-agatttctaggaattcaatcc-3'); mutant Stat5 (5'-agatttagtttaattcaatcc-3', identical to the above consensus Stat5 except for the ctagg->agttt substitution); and a 21 oligomer corresponding to region -918 to -898 of the ntcp promoter (5'-ttgtcattcttggaaaaataa-3'). The probes were radiolabeled with [{gamma} 32P]ATP, using polynucleotide kinase at the 5'-OH (blunt) ends. The labeled oligomers were gel purified on a 20% polyacrylamide gel and eluted in NET buffer (0.1 M NaCl, 1 mM EDTA, 1 mM Tris/HCl, pH 7.6). Nuclear extracts (15–30 µg protein) were incubated for 20 min at room temperature with 20 fmol (~100,000 cpm) of purified probe in a 30 µl reaction buffer containing 5 mM Tris/HCl, pH 7.9; 15 mM HEPES/KOH, pH 7.9; 0.08 M KCl; 3.5 mM MgCl2; 5 mM EDTA; 10% glycerol; 0.1% Tween 20; and 0.133 mg/ml poly(dI-dC):poly. Free probe and protein-bound probe were separated on a 5% polyacrylamide gel containing 2.5% glycerol and gel running buffer [Tris-glycine buffer (0.38 mM glycine, 2 mM EDTA, and 50 mM Tris) for the ß-casein Stat5 probe, and 0.25 x TBE (25 mM Tris, 25 mM boric acid, and 25 mM EDTA, pH 8) for the ntcp probe]. The gel was dried and exposed to x-ray film at -80 C for 1–2 d. In competition assays, 0.4 and 2 pmol (20- and 100-fold molar excess) of the specific unlabeled oligo were added to the binding reaction. For supershift studies, the nuclear extract was preincubated with 1 µg Stat5, Stat5a, or Stat5b polyclonal antibodies for 30 min at room temperature before the addition of labeled oligomer.

Northern blot analysis
Total RNA was prepared from hepatocytes using TRIzol reagent (Life Technologies, Inc.) according to the manufacturer’s instruction, separated (20 µg/lane) in a 1% agarose-formaldehyde 3-(N-morpholino)propanesulfonic acid gel, and transferred to a Duralon UV membrane. Hybridization and posthybridization washes were carried out as described (31). cDNA probes of ntcp were prepared as described (31). To correct for the variance in total RNA loading and transfer among the groups, a 28S rRNA oligoprobe (a single-stranded 26-mer oligonucleotide) was end-labeled with [{gamma}32P]ATP and hybridized. Signals were visualized using a Molecular Dynamics, Inc. (Sunnyvale, CA) PhosphorImager and quantitated using the ImageQuant software. The blots were also exposed to Bio-Max MR-2 film (Eastman Kodak Co., Rochester, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PRL, mPL-I, and GH induce nuclear translocation and tyrosine phosphorylation of Stats in primary rat hepatocytes
The activation of Stat proteins is a key event by which PRL, PLs, and GH regulate gene transcription (4, 15, 21). Upon stimulation of specific cell surface receptors with corresponding ligand, cytoplasmic Stat proteins such as Stat 1, -3, -5a, and -5b dimerize, translocate into the nucleus, and bind to specific genes to activate transcription (11, 12, 13, 15, 21). To determine whether these Stat proteins were activated in liver under near-physiological condition by these hormones, freshly isolated primary rat hepatocytes were exposed to various concentrations of oPRL, mPL-I, or rGH for 60 min, and nuclear extracts were prepared. To determine whether PRL induced tyrosine phosphorylation of Stat1, -3, -5a, or -5b and nuclear translocation of these activated forms, nuclear extracts were first immunoprecipitated with antiphosphotyrosine monoclonal antibody PY-20, and immunoprecipitates were probed individually with antibodies to Stat 1, -3, -5a, or -5b. oPRL stimulated tyrosine phosphorylation of Stat5a and Stat5b in a dose-dependent manner, and this activation was clearly evident when cells were treated with 0.1 µg/ml oPRL (Fig. 1AGo). The very faint band of Stat1 seen at 10 µg/ml oPRL concentration was variable, given that such a band was not visible when cells were treated with 100 µg/ml oPRL (data not shown). No activation of Stat3 was detected. Stat1 and Stat3 were found in the nucleus, in their unactivated forms, to the same extent in the presence and absence of PRL (data not shown). When added to hepatocytes isolated from pregnant rats, mPL-I was very effective in increasing nuclear translocation of tyrosine phosphorylated Stat5a, -5b, and -1 at both 0.5 and 1 µg/ml (Fig. 1BGo). Similarly, rGH, at concentrations as low as 0.01 µg/ml, stimulated tyrosine phosphorylation and nuclear translocation of Stat5a, -5b, and -1 in hepatocytes from control female rats (Fig. 1CGo). Neither mPL-I nor rGH stimulated tyrosine phosphorylation and nuclear translocation of Stat 3. IL-6, used as a positive control, did activate Stat 3 in hepatocytes (data not shown). The time course of Stat5a and -5b activation in hepatocytes by oPRL, rPRL, mPL-I, and rGH was also examined. As shown in Fig. 1Go, D–G, the tyrosine phosphorylation of Stat5a and -5b was detectable 5–10 min after the addition of ligand.



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  		Figure 1. The dose response and time course relationship of oPRL, mPL-I, and rGH-induced tyrosine phosphorylation of Stat proteins in primary rat hepatocytes. Freshly isolated rat hepatocytes were treated for 1 h with the indicated concentrations of oPRL (A), mPL-I (B), or rGH (C). The nuclear proteins were isolated and immunoprecipitated with antiphosphotyrosine antibody (PY-20) and the immunoprecipitate probed with the indicated antibodies by Western analysis. This figure also shows the time course of tyrosine phosphorylation of Stat5a and -5b from nuclear protein of hepatocytes treated with 1 µg/ml oPRL (D), rPRL (E), mPL-I (F), or rGH (G).

 
PRL, mPL-I, and GH lead to binding of Stat5 to the ß-casein promoter in primary rat hepatocytes
We next examined whether these hormones were able to enhance DNA binding activity of nuclear proteins to the ß-casein Stat5 response element in primary hepatocytes. As shown in Fig. 2Go, no binding to the oligomer was detected with nuclear protein from untreated hepatocytes. Stimulation of hepatocytes with oPRL, mPL-1, and rGH induced a single DNA-protein complex. This complex was supershifted by preincubation with anti-Stat5 antibody, indicating the presence of Stat5 in the complex. Characterization of the time course of binding, by EMSA, revealed that DNA binding activity occurred in parallel with the tyrosine phosphorylation of Stat5 (data not shown).



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  		Figure 2. EMSAs showing induction of DNA binding activity of nuclear proteins by oPRL, mPL-I, and rGH in primary rat hepatocytes. Nuclear proteins were extracted from freshly isolated rat hepatocytes treated with medium or 1 µg/ml oPRL, mPL-I, or rGH and incubated with the ß-casein 32P-labeled Stat5 consensus oligonucleotide. Polyclonal rabbit anti-Stat5 (1 µg) supershifted the DNA/protein complexes, indicating the presence of Stat5. Ab, Antibody; SS, supershift.

 
Further competition and antibody supershift assays, using nuclear protein extracts from oPRL-treated hepatocytes, were conducted to identify the characteristics of the binding. As seen in Fig. 3Go, PRL induced the DNA-protein complex in a dose-dependent manner (lane 1–3), and the specificity of the binding was verified by the inhibition of complex formation by the addition of 20- and 100-fold excess of the unlabeled Stat5 consensus oligo (lanes 4 and 5 vs. 3) but not by the mutant Stat5 consensus oligo (lanes 6 and 7). In addition, this specific protein-DNA complex formation was also decreased by 20- and 100-fold excess of an unlabeled 29 oligomer corresponding to region -922 to -892 of the ntcp promoter encompassing one of the Stat5 elements (lanes 8 and 9). These DNA-protein complexes were strongly supershifted by anti-Stat5b antibody and partly by anti-Stat5a antibody (Fig. 3Go, lanes 10 and 11).



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  		Figure 3. EMSA competition assays of PRL-induced DNA binding activity of nuclear proteins in isolated hepatocytes. Nuclear extracts prepared from hepatocytes treated with oPRL were incubated with 32P-labeled Stat5 consensus binding sequence from the ß-casein promoter. In competition assays, a 20- and 100-fold molar excess of unlabeled Stat5 consensus (Stat5, lanes 4 and 5), mutant Stat5 (Mstat 5, lanes 6 and 7), and ntcp (Ntcp, lanes 8 and 9) oligo was used. The supershift assays (lanes 10 and 11) were conducted by using polyclonal rabbit anti-Stat5a and anti-Stat5b, respectively.

 
PRL enhances the binding of Stat5a and Stat5b to the ntcp gene promoter
Two Stat5 consensus sequences have been identified within the immediate 1.3-kb 5' flanking region of the ntcp promoter, with which sequence-specific regulatory proteins could interact and potentially regulate its transcription. A 21 oligomer containing the Stat5 response element located -912 to -904 (TTC TTG GAA) in the promoter of ntcp was employed to evaluate its ability to bind nuclear proteins. As shown in Fig. 4Go, a clear DNA-protein complex was observed in the presence of PRL (lanes 1–3), in agreement with our previous in vivo studies (34). The formation of this complex was inhibited by a 20- and 100-fold molar excess of unlabeled ntcp oligomers (lanes 4, 5) and consensus Stat5 oligomers (lanes 6, 7), but not by the mutant Stat5 oligomers (lanes 8, 9), verifying the specificity of the binding. Additionally, the complex was markedly supershifted with Stat5b antibody (lane 11) and weakly shifted with Stat5a antibody (lane 10), confirming the presence of Stat5b, and possibly of Stat5a, in the complex.



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  		Figure 4. EMSA competition assays of PRL-induced DNA binding activity of nuclear proteins toward the Stat5 response element in the ntcp promoter. Nuclear extracts prepared from hepatocytes treated with oPRL were incubated with a 32P-labeled 21 oligomer corresponding to -918 to -898 of the ntcp promoter. In competition assays, a 20- and 100-fold molar excess of Ntcp (lanes 4 and 5), Stat5 (Stat5, lanes 6 and 7), and Mstat5 (lanes 8 and 9) oligomer were used. The supershift assays (lanes 10 and 11) were conducted using polyclonal rabbit anti-Stat5a and anti-Stat5b, respectively.

 
PRL, PL-I, and GH increase mRNA expression of ntcp in freshly isolated rat hepatocytes
We next examined whether PRL, PL, or GH treatment was able to induce ntcp gene expression in hepatocytes, which would reflect the effects of these hormones under physiological conditions. Because expression of PLs is unique to pregnancy, hepatocytes from pregnant rats were used for mPL-I treatment. Freshly isolated hepatocytes were treated with increasing concentrations of oPRL, mPL-I, or rGH for 2 h; after which, total RNA was isolated and subjected to Northern analysis (Fig. 5Go); 28s RNA was used as control. Treatment of hepatocytes with PRL, mPL-I, or rGH significantly increased ntcp mRNA expression in a dose-dependent manner (Fig. 5Go). Quantitative analysis of Northern blot by phosphorimaging indicated a 4-fold increase in ntcp mRNA after PRL and an approximate 5-fold increase after mPL-I or rGH treatment.



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  		Figure 5. Induction of ntcp mRNA by oPRL (A), mPL-I (B), and rGH (C) in primary rat hepatocytes. Freshly isolated hepatocytes from normal female (A and C) or pregnant (B) rats were treated with the indicated concentrations of oPRL, mPL-I, or rGH for 2 h. Total RNA was isolated and used for Northern analysis to determine ntcp mRNA expression. Results were quantified by phosphorimager, standardized against controls, and mathematically adjusted to yield a unit of 1 for the RNA from hepatocytes treated with blank medium. Fold inductions are shown above each lane. Because experiments were performed independently, different backgrounds were seen among experiments. The same results were reproduced with total RNA in hepatocytes from three independent animals.

 
Both PRL activation of Stat5 and induction of ntcp mRNA are inhibited by the protein tyrosine kinase inhibitor genistein
To explore further the relationship between tyrosine phosphorylation of Stat5 and the expression of ntcp mRNA in hepatocytes, we examined the action of the specific tyrosine kinase inhibitor, genistein, on PRL activation (Fig. 6Go). Freshly isolated hepatocytes were pretreated with 20 (lane 3), 50 (lane 4), or 100 µM (lane 5) genistein for 60 min before the addition of PRL. Genistein inhibited PRL-induced Stat5a and Stat5b phosphorylation and PRL-induced ntcp mRNA expression, supporting the hypothesis that tyrosine phosphorylation of Stat 5 is involved in the induction of ntcp gene expression.



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  		Figure 6. Inhibition of PRL-induced tyrosine phosphorylation of Stat5 and ntcp mRNA expression in primary rat hepatocytes. Freshly isolated rat hepatocytes were preincubated with medium or genistein (20, 50, or 100 µM) for 1 h, before the addition of PRL (10 µg/ml). After another 1 (A) or 2 h (B) incubation with PRL, nuclear extracts and total RNA were isolated for Western or Northern analysis. A, Nuclear extracts immunoprecipitated with PY-20 were probed with antibodies to Stat5a and Stat5b; B, Northern analysis for detecting expression of ntcp mRNA. The same results were reproduced with total RNA in hepatocytes from three independent animals. IP, immunoprecipitation.

 
mPL-I and GH, like PRL, transactivate the ntcp promoter in HepG2 cells
We further examined the signal transduction pathway of PRL, mPL-I, and GH by conducting transfection experiments in HepG2 cells. Our previous studies showed that oPRL acts via the rPRLRL and Stat5 to transactivate the ntcp luciferase reporter construct in HepG2 cells (34). The present investigation was designed to determine whether the PLs, represented by mPL-I, and GH could also act via the rPRLRL or rGHR to increase ntcp gene transcription. HepG2 cells were cotransfected with expression vectors for Stat5a, rPRLRL, or rGHR or mGHR, and the luciferase reporter construct with the ntcp minimal promoter (4 x 0.2pGL3), the native ntcp promoter p1237Luc, or the ntcp promoter with the native Stat5 response elements linked to the thymidine kinase promoter (0.5pT109Luc), and treated with the different hormones. mPL-I acted via the rPRLRL to increase the expression of the ntcp minimal promoter and was somewhat more effective than oPRL (Fig. 7AGo). Cotransfection with both rPRLRL and Stat5a was essential for mPL-I to transactivate the ntcp promoter (Fig. 7BGo), as noted previously for oPRL (34). mPL-I could not increase ntcp transcription in HepG2 cells transfected with the rGHR or the mGHR at 0.5, 1, 5, and 10 µg/ml (data not shown). Nuclear translocation of phosphorylated Stat5a was observed in response to both oPRL and mPL-I stimulation in transfected HepG2 cells (Fig. 7CGo), providing further evidence of the role of Stat5a in signal transduction. mPL-I and oPRL were equally effective in transactivating both the native ntcp promoter (p-1237Luc, Fig. 7EGo) and the native promoter linked to the thymidine kinase promoter (0.5pT109Luc, Fig. 7DGo). In addition, rPRL and oPRL showed a similar potency in transactivating the ntcp minimal promoter (Fig. 7FGo).



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  		Figure 7. Transactivation of the ntcp gene by oPRL, mPL-I, rGH, or hGH in HepG2 cells. HepG2 cells were cotransfected with expression vectors for Stat5a, rPRLRL, or rGHR, and the luciferase reporter construct with the ntcp minimal promoter coupled to 4 Stat5 response elements (4 x 0.2pGL3), the ntcp promoter containing the two native Stat5 response elements linked to the thymidine kinase promoter (0.5pT109Luc), or the native ntcp promoter p1237Luc. ß-Galactosidase was included for normalization of data. Cells were treated with hormones and luciferase activity assayed, or nuclear extracts isolated for Western analysis. Results represent the mean ± SEM of three independent experiments. A, oPRL and mPL-I transactivated 4 x 0.2pGL3 to a comparable extent in HepG2 cells cotransfected with rPRLRL and Stat5a. B, mPL-I induced transactivation of 4 x 0.2pGL3 was observed only in cells cotransfected with both the Stat5a and PRLRL plasmids. C, Tyrosine phosphorylation of Stat5a by oPRL or mPL-I in HepG2 cells cotransfected with PRLRL and Stat5a. Nuclear extracts from unstimulated and hormone-stimulated transfected cells were immunoprecipitated with PY-20 and immunoblotted with mouse monoclonal anti-Stat5 antibody. D, mPL-I and oPRL induced comparable transactivation of the native Stat5 response elements in 0.5pT109luc. E, mPL-I and oPRL induced comparable transactivation of the native ntcp promoter p1237luc. F, oPRL and rPRL showed similar transactivation of 4 x 0.2pGL3. G, rGH and hGH, but not oPRL, transactivated 4 x 0.2pGL3 via the cotransfected rGHR and Stat5a in a dose-dependent manner. H, oPRL and hGH, but not rGH, transactivated 4 x 0.2pGL3 via the cotransfected rPRLRL and Stat5a. oPRL and hGH were equally potent in inducing the Stat5 responsive reporter gene construct.

 
As shown in Fig. 7GGo, both rGH and hGH transactivated the ntcp gene via the rGHR. hGH was also able to increase the expression of the ntcp promoter via the rPRLRL (Fig. 7HGo). On the other hand, rGH did not increase the bile acid transporter gene expression via rPRLRL; and conversely, oPRL did not transactivate via the rGHR. Cotransfection of both GHR and Stat5 was critical for the transactivation of ntcp by rGH (data not shown).

Like PRL, mPL-I requires PRLRL to transactivate ntcp gene expression in HepG2 cells, and cotransfection with GHR decreases the activity
Though studies in HepG2 cells showed that oPRL is not able to transduce the signal through PRLRS (34), we questioned whether mPL-I might act via PRLRS. HepG2 cells were cotransfected with expression vectors for Stat5a, rPRLRL or rPRLRS, and the luciferase reporter construct containing the ntcp minimal promoter (4 x 0.2pGL3), and treated with rPRL or mPL-I. Neither rPRL nor mPL-I transactivated the ntcp promoter via rPRLRS (Fig. 8AGo). Recent studies have demonstrated that oPL is capable of functional heterodimerization of oGHR and oPRLR in transfected 293-HEK cells and that this heterodimerization increases oPL-induced signal transduction (47). We therefore examined the potential role of heterodimerization of rPRLRL and rGHR in mPL-I signaling in liver cells. HepG2 cells were cotransfected with expression vectors for Stat5a, rPRLRL, rGHR, or rPRLRL plus rGHR (1:1 ratio) or rPRLRL plus rGHR (1:4 ratio), and the luciferase reporter construct with the ntcp minimal promoter (4 x 0.2pGL3), and treated with mPL-I. As shown in Fig. 8BGo, cotransfection with both rGHR and rPRLRL decreased luciferase activity, relative to that observed in cells transfected with PRLRL alone. Cotransfection with rGHR and rPRLRL in a 4:1 ratio showed less activity than cotransfection with rGHR and rPRLRL in a 1:1 ratio. These findings indicate that in transiently cotransfected HepG2 cells, under conditions where heterodimers of rGHR and rPRLRL could form, the rPRLRL homodimers were most effective in transducing the signal by these hormones.



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  		Figure 8. rPRL and mPL-I transactivate the ntcp promoter via PRLRL, but not PRLRS or GHR. HepG2 cells were cotransfected with Stat5a, 4 x 0.2pGL3, rPRLRL or rPRLRS (A), or rPRLRL, rPRLRL plus rGHR, or rGHR (B) as indicated. The different proportion of rPRLRL and rGHR was based on micrograms of DNA. ß-galactosidase was included for normalization of data. Cells were treated with the indicated concentrations of hormones, and luciferase activity was assayed. Results represent the mean ± SEM of three independent experiments.

 
PRL and GH lead to binding of Stat5 to the ß-casein promoter in transfected HepG2 cells
In parallel with the luciferase assays in HepG2 cells transfected with the rGHR or rPRLRL and Stat5a, gel shift assays were performed using a 21 oligomer from the bovine ß-casein promoter containing the Stat5 consensus binding sequence to establish the role of Stat5 in signal transduction. As shown in Fig. 9Go, upon exclusion of the rPRLRL (lanes 1, 3, and 4) or Stat5a (lanes 1–3) from the cotransfection experiment, no signal could be observed when the nuclear extracts were probed with the radiolabeled consensus Stat5 oligonucleotide, relative to the strong signal observed when cells transfected with rPRLRL and Stat5a were stimulated with oPRL (lane 5). A 50-fold molar excess of the unlabeled oligomer decreased the signal significantly (lane 6 vs. lane 5), while preincubation with an antibody to Stat5a led to a supershift complex (lane 12), confirming the role of Stat5 in signal transduction. Concordant with the luciferase assays, rGH acted via the rGHR, but not the rPRLRL, to activate Stat5a (lane 8 vs. lane 10) and oPRL did not activate Stat5a via the rGHR (lane 9). The hGH induced nuclear translocation of phosphorylated STAT5a via both the rPRLRL (lane 7) and rGHR (lane 11).



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  		Figure 9. EMSAs showing induction of DNA binding activity of nuclear proteins by oPRL, rGH, and hGH in transfected HepG2 cells. HepG2 cells were cotransfected with expression vectors for Stat5a, and rGHR or rPRLRL, as indicated. The transfected HepG2 cells were exposed to 0.5 µg/ml oPRL, rGH, or hGH for 30 min, and nuclear extracts were prepared and subjected to EMSA using a 32P-labeled Stat5 consensus oligonucleotide from the bovine ß-casein gene promoter. Oligo, oligomer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study clearly establishes the mechanisms by which PRL, GH, and PL activate transcription of the ntcp gene, and it provides strong evidence for hormonal regulation of ntcp expression during different physiological conditions, such as pregnancy and lactation in the postpartum period. Our previous study showed PRL responsiveness of the ntcp promoter activity in transiently transfected HepG2 cells by activation of Stat5 via PRLRL (34). However, the mechanisms obtained from studies in cell lines, where the receptors and their transducer proteins are usually over-expressed, cannot alone ensure a similar mechanism of these events in vivo or under normal physiological conditions. Thus, the relative levels of expression of receptors and transcription factors vary, depending on species, age, physiological status, tissues, and other factors. Moreover, different receptor isoforms (e.g. rPRLRL and rPRLRS in liver) exist in vivo and therefore may modulate the ability to transduce the hormonal signals. We therefore examined the effects of PRL, PLs, and GH under near-normal physiological conditions in freshly isolated hepatocytes.

The binding of GH and PRL to their respective receptors has been shown to activate Stat1, -3, -5a, and -5b (16, 20); transcriptional regulation by these activated Stat proteins is a key event in signaling by these hormones. Stat5 phosphorylation in response to PL-I has also been reported (21). Therefore, we first examined the activation of tyrosine phosphorylation of these distinct Stat proteins in response to oPRL, mPL-I, and rGH in primary rat hepatocytes. oPRL activated both Stat5a and Stat5b by tyrosine phosphorylation and facilitated their translocation to the nucleus (Fig. 1AGo). In contrast, tyrosine phosphorylation of Stat1 and Stat3 by PRL could not be detected (Fig. 1AGo), although high constitutive levels of unactivated Stat1 and Stat3 were found in the nucleus of hepatocytes (data not shown). PRL was able to induce activation of Stat5 at concentrations as low as 0.1 µg/ml, which are comparable with PRL serum levels in postpartum suckling rats. These data directly demonstrate, for the first time, the activation of Stat5 by PRL in hepatocytes, and strongly support the hypothesis that PRL secreted by postpartum suckling rats is able to activate Stat5a and Stat5b in the liver. Consistent with previous reports (16, 48), GH induced tyrosine phosphorylation of Stat5a, -5b, and Stat1 in hepatocytes (Fig. 1CGo). This study also provides the first evidence for PL-induced tyrosine phosphorylation of Stat5a, -5b, and Stat1 in hepatocytes. The basis for activation of Stat1 by mPL-I in hepatocytes from pregnant rats is not known, but it may reflect the increase in expression of the PRLRL described in pregnancy (22). No activation of Stat3 was detected by any of these three hormones.

The activation of Stat5 proteins by PRL, PL, and GH was also confirmed by the gel mobility shifts assays, where the DNA binding activity of nuclear protein extracts to the Stat5 response element in the bovine ß-casein promoter was induced in primary hepatocytes (Fig. 2Go). Competition and supershift assays showed that activated Stat5b, and probably Stat5a, were able to bind to the Stat5 response element in the bovine ß-casein promoter as well as to the element of the ntcp promoter in hepatocytes treated with PRL (Figs. 3Go and 4Go). The supershift assay revealed that Stat5b protein was much more abundant than Stat5a in the DNA-protein complexes (Figs. 3Go and 4Go), in agreement with previous findings that Stat5b is the major Stat5 isoform in the liver (16). Stat5a and Stat5b are the products of two different genes that are nearly identical (49). They differ most markedly in their carboxyltermini, which are involved in transcriptional activation (50). In this regard, defects observed in the knockout models of these two isoforms differ (51, 52), which indicates that Stat 5a and Stat5b are not completely redundant and may activate different subsets of genes. The current data suggest that Stat5b is the major isoform involved in PRL-induced transactivation of ntcp. However, in HepG2 cell cotransfection assays, ovine Stat5a also transactivates ntcp, suggesting that both Stat5a and Stat5b are able to transactivate ntcp in liver. Therefore, in the case of the ntcp gene, the complexes may be composed of Stat5a or Stat5b homodimers and/or Stat5a-5b heterodimers. Further studies will be needed to establish definitively the role of Stat5a vs. Stat5b in the activation of ntcp.

Studies of the time course of Stat5a/5b activation by rPRL, oPRL, rGH, and mPL-I in hepatocytes detected tyrosine phosphorylation of Stat5 within 10 min (Fig. 2Go, D–G), consistent with studies in Nb cells (10) and COS cells (11), where the activation of Stat5 occurs within 10 min after the addition of PRL. However, the time of detection of phosphorylated Stat5 varied among different preparations and hepatocytes from different animals, ranging from 5–20 min.

We next directly examined the effect of these three hormones on the expression of ntcp mRNA in hepatocytes. PRL, PL, and GH increased ntcp mRNA within 2 h (Fig. 5Go). These data indicate that once Stat5 binds to the ntcp promoter, the transcription of the gene was quickly activated. Pretreatment of the hepatocytes with the tyrosine kinase inhibitor genistein prevented PRL-induced Stat5 activation (Fig. 6AGo) as well as the increase in expression of ntcp mRNA (Fig. 6BGo). These data suggest that in hepatocytes, PRL-induced ntcp gene expression requires the activation of Stat5a and/or Stat5b by tyrosine phosphorylation. Genistein may produce this effect by inhibition of tyrosine phosphorylation of PRLRL or Jak2 as well.

The present studies are consistent with several lines of evidence indicating that PRL is responsible for the increased hepatic expression of ntcp mRNA and Na+/TC cotransport activity observed postpartum. Early studies showed that treatment of rats with bromocriptine, which decreases PRL released by the suckling stimulus, prevents the increase in Na+/TC cotransport activity and ntcp mRNA observed postpartum (31, 32). In addition, iv infusion of oPRL (300 µg/d) for 7 d, to ovariectomized rats, increases Na+/TC cotransport activity and ntcp mRNA (31). Finally, the suckling stimulus, which increases serum PRL levels up to 700 ng/ml, induces nuclear translocation of the phosphorylated Stat5 that binds to the Stat5 response elements in the ntcp promoter (34). In agreement with these early studies, the present studies further demonstrated that PRL, as well as PL and GH, activated both Stat5a and Stat5b and facilitated their translocation to the nucleus, where they bound to specific DNA sequences in the promoter of ntcp and activated its transcription.

Jahn et al. (37) reported that a single injection (it is not clear whether oPRL was administered sc, as stated in the text, or ip, as stated in the legends) of 250 µg oPRL to ovariohysterectomized rats, on d 19 of pregnancy, activates the Jak2/Stat5 pathway in mammary gland but not in the liver. In addition, Choi et al. (36) demonstrated the presence of a low level of Stat5 DNA-binding activity in adult female rat liver but concluded that PRL is not responsible for this low-level Stat5 activity because activation of Stat5 did not correlate with PRL levels during the estrus cycle. Treatment of normal female rats with bromocriptine, to inhibit PRL secretion, did not affect Stat5 activation, nor did direct treatment with rPRL (12.5 or 50 µg/100 g, ip), leading these authors to conclude that PRL does not activate Stat 5 in liver. However, the present study clearly demonstrates that in freshly isolated hepatocytes, PRL transduced its signal to Stat5a and Stat5b and enhanced the expression of ntcp. The question therefore remains as to why PRL is able to activate Stat5 in isolated hepatocytes but not after ip/sc administration in vivo. It is likely that the presence of the dominant PRLRS in liver blocks the activation of Stat5a/5b by PRL at lower concentrations or during a short duration of exposure. It is possible, therefore, that isolation of rat hepatocytes has led to the selective sequestration or inactivation of the PRLRS or modification of the PRLRL, and thus increased the potency of PRL in culture. Alternatively, and in the absence of data quantitating hepatic exposure to PRL administered ip/sc, it is possible that the delivery and duration of exposure to effective concentrations of PRL in liver in this model is inadequate to activate Stat 5. Further studies in vivo or in the perfused liver are necessary to resolve this issue. With some reserve, therefore, we advance the view that PRL is responsible for the high level of activation of Stat5 and increased ntcp mRNA and Na+/TC cotransport in the lactating postpartum rat, where the serum PRL is dramatically elevated.

The present study further examined the signal transduction pathways by PL and GH by using the transiently transfected HepG2 cells. GH and PL, like PRL, were also able to transactivate the ntcp promoter via the corresponding receptors and Stat5 protein (Fig. 7Go). This study is the first to demonstrate the transcriptional regulation of the bile acid transporter ntcp by PLs and GH. The greater potency of GH in activating Stat5a/5b may reflect the higher concentration of GHR in hepatocytes. This is consistent with the previous findings that a single injection of GH (3–150 µg/100 g, ip) induces the activation of liver Stat proteins in vivo (16). The mechanism of PL induction closely resembles that by PRL, in that PL activates ntcp gene transcription via the rPRLRL but not the GHR or rPRLRS. As anticipated, rGH acted only via the GHR, whereas hGH activated ntcp transcription via both the GHR and the PRLRL (Fig. 7Go, G and H), consistent with the somatogenic and lactogenic activities of primate GH (1). The nuclear translocation of phosphorylated Stat5a, in response to oPRL, mPL-I, and rGH observed in transfected HepG2 cells, is consistent with findings in other systems (21, 53, 54, 55). These data strongly suggest that PRL and mPL-I activate Stat5a/5b and increase ntcp expression in hepatocytes through the PRLRL. Cotransfection of HepG2 cells with rGHR and rPRLRL decreased luciferase activity induced by mPL-I, relative to that seen when only the rPRLRL was cotransfected with Stat5a, suggesting that the heterodimerization of rGHR and rPRLRL was not as effective as the homodimerization of rPRLRL in transducing mPL-I signals. In contrast, Herman et al. (47) found that cotransfection of the oGHR and oPRLRL greatly augmented the activity observed in cells transfected with oPRLRL alone. Although the potential for cross-talk between PRLRL and GHR in signal transduction by PRL or mPL-I deserves to be further addressed, the present studies support the hypothesis that PRL and mPL-I act via the PRLRL alone.

In summary, the present data establish that PRL, PL, and GH induce ntcp gene expression via activation of Stat5 proteins. The phosphorylated Stat5a/5b were translocated to the nucleus, where they bound to specific DNA sequences in the promoter of ntcp and activated its transcription. Our finding that these three hormones increase the expression of ntcp, sharing common factors and mechanism, highlights the importance of maintaining this key liver function under various physiologic conditions. Under normal conditions, the basal and tissue-restricted expression of ntcp may be directed by transcription factors such as a TATA element, hepatocyte nuclear factor 1, and retinoic acid receptor/retinoid X receptor (28, 56). The down-regulation of ntcp, by suppressed nuclear levels of hepatocyte nuclear factor 1 and retinoic acid receptor/retinoid X receptor during liver inflammation, underscores the importance of these transcriptional factors (56). In lactation, the elevated PRL is primarily responsible for the increased expression of ntcp; whereas, during pregnancy, PLs may be critical for maintaining ntcp expression in the face of suppressed levels of PRL and elevated levels of estrogens, which, in high doses, have been shown to suppress ntcp expression and activity (40, 57). We have recently reported that expression of bsep, which mediates the ATP-dependent transport of bile salts from the hepatocytes into bile, is also up-regulated postpartum, and by infusion of PRL, indicating a coordinate up-regulation of both hepatic bile salt transporters by PRL (33). These findings, together with the marked hypertrophy of the small intestine in postpartum rats (58) and increased expression of the intestinal apical sodium-dependent bile salt transporter (59), imply an important PRL-mediated regulation of metabolic function by increasing the enterohepatic circulation of bile acids during lactation. Increased enterohepatic circulation of bile salts may serve to meet the high nutritional needs of the lactating dams to increase absorption of fats and fat-soluble vitamins. Incorporation of these fats and fat-soluble vitamins into milk would also serve to provide these essential nutrients to the developing pups.


   Footnotes
 
This work was supported by Training Grant ES 07266 (to M.W.) and PHS Grant DK-46923.

Abbreviations: bsep, Bile salt export pump; Jak2, Janus kinase 2; mPL, mouse placental lactogen; ntcp, Na+/TC cotransporting polypeptide; oPRL, ovine PRL; PL, placental lactogen; PMSF, phenylmethylsulfonylfluoride; PRLR, PRL receptor; PY-20, antiphosphotyrosine antibody; PY-20AC, phosphotyrosine agarose conjugated; rGH, rat GH; rGHR, rGHR receptor; rPRLRL, long form of the rat PRL receptor; rPRLRS, short form of the rat PRL receptor; Stat, signal transducer and activator of transcription; TC, taurocholate; WE medium, Williams E medium.

Received March 5, 2001.

Accepted for publication June 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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