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Endocrinology Vol. 145, No. 4 1739-1749
Copyright © 2004 by The Endocrine Society

Estradiol Represses Prolactin-Induced Expression of Na+/Taurocholate Cotransporting Polypeptide in Liver Cells through Estrogen Receptor-{alpha} and Signal Transducers and Activators of Transcription 5a

Jingsong Cao, Marcie Wood, Yong Liu, Tim Hoffman, Jim Hyde, Ok-Kyong Park-Sarge and Mary Vore

Graduate Center for Toxicology, Departments of Physiology and Anatomy and Neurobiology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0305

Address all correspondence and requests for reprints to: Mary Vore, Ph.D., H.S.R.B 306, Graduate Center for Toxicology, University of Kentucky, 800 Rose Street, Lexington, Kentucky 40536-0305. E-mail: maryv{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Na+/taurocholate cotransporting polypeptide (ntcp) mediates the uptake of bile salts from plasma across the basolateral domain of the hepatocyte. We have demonstrated that ntcp expression can be induced by prolactin (PRL) and placental lactogen via the PRL receptor and signal transducers and activators of transcription (Stat)5a pathway. However, elevated levels of placental lactogen do not increase the expression of ntcp in pregnant rats. Because plasma estradiol (E2) levels are also elevated in pregnancy, we investigated the inhibitory effects of E2 on PRL-induced ntcp activation. E2 treatment inhibited the PRL-induced increase in liver ntcp mRNA to the same levels as in rats treated with E2 alone. Estrogen receptor-{alpha} (ER{alpha}) mRNA and protein expression in liver were increased 2.6-fold and 2.2-fold, respectively, in pregnancy relative to controls. In HepG2 cells, E2 repressed PRL-induced ntcp reporter gene expression in a dose-dependent manner in the presence of cotransfected ER{alpha}. The ER{alpha} antagonist ICI 182,780 reversed E2-induced repression, indicating specificity of inhibition by E2. Overexpression of coactivator p300 did not reverse the inhibitory effects of E2 and ER{alpha}. Western and gel shift analysis revealed that E2-bound ER{alpha} decreased the tyrosine phosphorylation and DNA-binding activity of Stat5a, indicating that the inhibitory effect of E2 was mediated, at least in part, by interfering with PRL-mediated signal transduction. The present studies demonstrate the physiological significance of cross-talk between ER{alpha} and Stat5a in liver, in which both proteins are expressed. These data also establish a novel mechanism by which expression of ntcp, an important hepatic bile acid transporter, can be regulated by multiple hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FORMATION OF bile is a vital function of liver and is essential for excretion of cholesterol and metabolites of endogenous and exogenous products. The active transport of osmotically active solutes from plasma to the confined space of the canaliculus, followed by the passive movement of water, provides the basis of bile formation (1). Bile salts are the most abundant solutes in bile and participate in the emulsification and absorption of fats, steroids, and fat-soluble vitamins and xenobiotics. Na+/taurocholate cotransporting polypeptide (ntcp) is the major transporter responsible for the uptake of bile salts from plasma across the basolateral domain of the hepatocyte into the liver (2). The rat ntcp gene has been characterized and several elements that direct its basal and tissue-restricted expression, including hepatocyte nuclear factor-1 and retinoid response elements, have been identified within the minimal promoter (3). Prolactin (PRL) plays a critical role in increasing maternal bile secretory function and increasing Na+/taurocholate cotransport activity by increasing ntcp mRNA and protein expression in postpartum lactating rats (4, 5, 6, 7). We recently demonstrated that PRL, placental lactogen (PL), and GH induce ntcp expression via activating signal transducers and activators of transcription (Stat)5 in freshly isolated hepatocytes as well as in transiently transfected HepG2 cells (8). PRL and PL activate Stat5a/b via the long form of the PRL receptor (PRLRL), whereas GH activates Stat5a/b via the GH receptor. The binding of hormone ligand to the respective receptor leads to receptor dimerization and activation of associated Janus kinase 2 that phosphorylate a tyrosine in the cytoplasmic domain of the receptor, followed by recruitment and tyrosine phosphorylation of the Stat5a/b transcription factors. The phosphorylated Stat5a/b dissociate from the receptor, dimerize and are translocated to the nucleus, in which they bind to two specific DNA sequences ({gamma}-interferon activated sequences, TTCnnnGAA) located in the -973- to -904-bp region of the ntcp promoter and activate its transcription (8, 9). These studies established the molecular basis by which PL and PRL regulate liver metabolic function under different physiological conditions such as pregnancy and lactation, respectively.

In rodents, PRL secretion by the pituitary declines in pregnancy as levels of PL, secreted by trophoblast giant cells, increase (10, 11). However, both ntcp mRNA and protein levels are maintained in pregnancy relative to that in nonpregnant rats (6, 7), despite the demonstrated ability of PL to increase ntcp mRNA expression in isolated hepatocytes (8). Plasma estradiol (E2) levels are also elevated in pregnancy (12), and high doses of ethynylestradiol have been shown to decrease expression of ntcp mRNA and activity (13, 14), although the underlying mechanisms are not clearly understood. We therefore hypothesized that estrogens, which are elevated in late pregnancy, might act to block PL-induced transcription of ntcp.

Estrogens play important roles in regulating the growth, development, and differentiation of many reproductive tissues including the uterus, vagina, ovary, testis, prostate, and mammary gland (15). They also exert important effects on tissues not involved in reproduction, including bone, the cardiovascular system, and liver (16, 17, 18). The physiological actions of estrogens are mediated by estrogen receptor (ER)-{alpha} and -ß, belonging to the superfamily of steroid/nuclear receptors (19, 20, 21, 22). The signal transduction pathway of estrogen includes ligand binding to intracellular ER, followed by a conformational change of the receptor(s) to an activated form, dimerization, recruitment of transcriptional auxiliary factors, binding of activated ER to estrogen response elements (ERE) in the target gene promoter, and regulation of transcriptional activity of target genes in conjunction with other transcription factors bound to their cognate sites in the promoter (15, 18, 23, 24, 25). The liver appears to express predominantly ER{alpha} (26), and its expression is also under multihormonal regulation (17, 27). The hepatic ER also plays important roles in human liver disease and animal liver injury models (16, 28, 29, 30).

The glucocorticoid receptor, mineralocorticoid receptor, and progesteroid receptor all synergize with Stat5 in the induction of transcription from the ß-casein gene promoter, thus establishing a functional interaction between steroid/nuclear receptors and Stat-mediated signal transduction pathways (31, 32, 33). In addition, the functional interaction between ER and Stat3 signaling pathway forms the molecular basis for the inhibitory effect of estrogens on IL-6 function (34). Recently ER{alpha} and ERß were found to potently repress PRL-induced Stat5 transcriptional activity by a direct physical interaction with Stat5 (35). These findings led us to postulate cross-talk between the ER and PRL/PL signaling pathways, especially in liver, in which ER and Stat5 are both expressed. Such cross-talk would likely be most pronounced in pregnancy, when circulating E2 and PL are markedly elevated. We therefore determined whether estrogens could block the PRL-induced increase in ntcp mRNA expression and characterized the role of cross-talk between ER and Stat5 signal transduction pathways in liver.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
E2 (estradiol-17ß) was obtained from Sigma (St. Louis, MO) and ICI 182,780 from TOCRIS (Ballwin, MO). Ovine PRL (NIDDK-oPRL-21; AFP10692C) was provided by the National Institute of Diabetes and Digestive and Kidney Diseases, the National Hormone and Pituitary Program, and Dr. A. F. Parlow. Recombinant mPL-I was a kind gift from Dr. Frank Talamantes (University of California, Santa Cruz, CA). All other chemicals were of analytical grade and were from Life Technologies (Rockville, MD), Fisher Scientific (Pittsburgh, PA), or Sigma. Antibodies used in this study were obtained from Zymed Laboratories Inc. (South San Francisco, CA: polyclonal rabbit antimouse antibodies to Stat5a), Upstate Biotechnology [Lake Placid, NY: monoclonal mouse anti-phospho-Stat5a/b (Y694/Y699) antibody and monoclonal mouse anti-p300 antibody], Affinity BioReagents (Golden, CO: monoclonal mouse anti-PRL receptor antibody), Alexis Biochemicals (San Diego, CA: polyclonal rabbit anti-ER{alpha} antibody), and Amersham Corp. (Arlington Heights, IL: donkey antirabbit IgG and donkey antimouse IgG horseradish peroxidase conjugates).

Plasmids
The expression vector containing ovine Stat5a cDNA (pXM-Stat5a) and the cDNA for the rat PRLRL (pL3-PRLRL) were kindly provided by Dr. B. Groner (Institute for Experimental Cancer Research, Freiburg, Germany) (36) and Dr. Paul Kelly (Institut National de la Santé et de la Recherche Médicale, Paris, France), respectively, and were prepared as described (9). The ntcp luciferase reporter construct 4x 0.2pGL3 containing 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 (Fig. 1Go) (9). The expression vector encoding human ER{alpha} (pCMV5-hER{alpha}) and the estrogen-responsive reporter plasmid 4x ERE-TK-Luc, which contains four copies of ERE in front of a thymidine kinase promoter reporter construct, were a gift from Dr. Benita Katzenellenbogen (University of Illinois, Urbana-Champaign, IL). The ERß expression vector (pCMV5-rERß) was constructed by ligating the EcoR1-Xba fragment (2269 bp) of the rat ERß (22, 37) into the pCMV5 plasmid and the ligation junction confirmed by sequencing. Consequently, this expression plasmid contains the two in-frame ATG initiation codons for the short (485 aa) and long (530 aa) ERß. Human p300 (pCMVß-p300) was a gift from Dr. Richard Goodman (Vollum Institute, Portland, OR). Dr. Daniel Noonan (University of Lexington, Kentucky) provided the plasmid pRSV-ß-galactosidase. Plasmid DNA was extracted as described (38) and purified by CsCl gradient centrifugation; the Stat5a plasmid was further purified on a sucrose gradient.



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  		FIG. 1. Plasmid constructs of the rat ntcp promoter reporters used in transfection. Two Stat5 response elements are located between -936 and -904 in the native promoter sequence (p-1237Luc). The minimal rat ntcp promoter reporter (4x0.2pGL3) contains four copies of Stat5 response element and the minimal rat ntcp promoter (-158 to +47).

 
Animals
Female Sprague Dawley timed pregnant rats (19–21 d of pregnancy), random-cycling female control rats, and ovariectomized (OVX) rats (200–250 g) were obtained from Harlan Industries, Indianapolis, IN. The rats had free access to food and water and were maintained on a 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 National Institutes of Health Guide for the Care and Use of Laboratory Animals. OVX rats were treated with bromocriptine (two 7.5-mg pellets sc; 10-d release; Innovative Research of America, Toledo, OH) to block endogenous PRL secretion and divided into five groups: oil, E2, oPRL + oil, oPRL + E2, and solvent + E2. The oPRL (300 µg/d, 7 d) or solvent (SOL; 0.4 M NaHCO3, 1.6% glycerol, 0.02% sodium azide) was administered iv via osmotic minipump (Alzet 2001, Alza, Palo Alto, CA) attached to a catheter inserted into the jugular vein as described (5). E2 was dissolved in sesame oil (300 µg/ml) and administered via a 24-mm length of silastic tubing placed sc as described by Kalra and Kalra (39). Tubes filled with sesame oil alone (oil) served as a control. At the end of treatment, rat and ovine PRL (5) and E2 serum levels (40) were determined by RIA.

Cell culture and transfections
HepG2 cells were grown in DMEM/F12 (1:1) medium, supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 3.58 mM glutamine, 55 µg/ml gentamicin, and 1 µg/ml insulin (Invitrogen). A day before transfection, 2 million cells were subcultured into 100-mm plates in phenol-red-free DMEM supplemented with 10% charcoal stripped fetal bovine serum (Hyclone Laboratories, Logan, UT), glutamine, and gentamicin. Transfections were performed using Lipofectin reagent (Invitrogen) following the manufacturer’s instruction. Cells were transfected with 5 µg of ntcp luciferase construct 4x 0.2pGL3 or native ntcp promoter construct p1237Luc, 5 µg of pXM-Stat5a, and 1 µg of pL3-PRLRL. To monitor the estrogen-induced transcriptional activation, cells were transfected with 5 µg of the estrogen-responsive reporter plasmid 4x ERE-TK-Luc, 5 µg of pCMV5-hER{alpha} or pCMV5-rERß, or 5 µg of empty vector pCMV5 as negative control. In addition, cotransfections were performed using different amounts of pCMV5-ER{alpha} as indicated in the figure legend. In some experiments, 5 µg of pCMVß-p300 or its empty vector was cotransfected. Plasmid pRSV-ß-gal (5 µg) was included to monitor the transfection efficiency. The total DNA transfected was adjusted to 20 or 25 µg with empty vector DNA. Five hours after transfection, the medium was removed and the cells washed twice with PBS and replated in a 96-well plate. The cells were treated with blank medium or the indicated concentrations of ligands (oPRL, E2, or oPRL + E2).

For ER antagonist studies, cells were pretreated with the indicated concentrations of the antagonist ICI 182,780 for 2 h. After a further incubation with the indicated hormones for 36–48 h, cells were washed with PBS and lysed with 50 µl of lysis buffer for 20 min at room temperature. The cell extract (20 µl) was combined with 100 µl of 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, Hercules, CA). The composition of all the solutions and buffers used in the assays were as reported (9). The normalized luciferase response was calculated as relative light units/ß-galactosidase activity (A at 415 nm)/min. Each ligand treatment was conducted in duplicate at minimum and the mean 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. For Western analysis, the medium was removed 5 h after transfection, the cells washed twice with PBS, and fresh medium added. After incubation for an additional 36–48 h, cells were washed with PBS and harvested for preparation of whole-cell extract.

Northern blot analysis
Total RNA was prepared from rat livers according to Chomczynski and Sacchi (41). Northern blot analysis was performed to detect the expression of hepatic ntcp mRNA in response to oPRL and E2 treatment, using 20 µg total RNA, as described (8).

Real-time PCR
The expression of ER{alpha} and ERß mRNA in normal and pregnant rat liver was determined by real-time PCR analysis as described (42). Two micrograms of total RNA isolated as described above was used for first-strand cDNA synthesis using SuperScript preamplification system (Invitrogen) according to the manufacturer’s instructions in a volume of 20 µl. Real-time quantitative PCR was performed on the cDNA sample using the Lightcycler system (Roche Diagnostics, Indianapolis, IN). The following primer pairs, resulting in amplified products of 344 and 262 bp, respectively (26), were used: ER{alpha}, 5'-AATTCTGACAATCGACGCCAG-3' and 5'-GTGCTTCAACATTCTCCCTCCTC-3'; ERß, 5'-TTCCCGGCAGCACCAGTAACC-3' and 5'-TCCCTCTTTGCGTTTGGACTA-3'. The oligonucleotides used for the amplification of ß-actin mRNA were as previously described (42).

Western blot analysis
HepG2 cells were harvested and lysed for 30 min at 4 C in lysis buffer [50 mM Tris-HCl (pH 7.4); 150 mM NaCl; 1% Nonidet P-40; 0.25% sodium deoxycholate; 1 mM EGTA; 1 mM phenylmethylsulfonyl fluoride; 1 mM sodium orthovanadate; 1 mM NaF; and 5 µg/ml each of aprotinin, leupeptin, and pepstatin A], followed by removal of insoluble materials by centrifugation at 15,000 x g for 10 min at 4 C. Soluble supernatant of whole rat liver homogenate was prepared as described (42) and 100 µg of liver homogenate (for ER{alpha} protein analysis), 20 µg (for p300), or 80–100 µg (for Stat5a, PRLR) of HepG2 cell protein resolved on an 8.5% SDS-PAGE and 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-Stat5a or anti-phospho-Stat5a/b, 1 µg/ml anti-ER{alpha}, anti-PRLR, or anti-p300) dissolved in the same buffer for 1–2 h at room temperature. After four washes (5 min each), the membranes were incubated with antirabbit or antimouse 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 Corp.) for 5 min and exposed to x-ray film for 1–30 min.

EMSAs
The EMSAs were conducted as described (8, 9). Two double-stranded oligonucleotides were used (sense strand sequence; protein-binding sites are underlined): a 21 mer containing the bovine ß-casein Stat5 consensus binding motif (5'-agatttctaggaattcaatcc-3'), and a 21 mer corresponding to region -918 to -898 of the ntcp promotor (5'-ttgtcattcttggaaaaataa-3'). The probes were radiolabeled with [{gamma}32P]ATP and purified as described (8). Cell extracts (100 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(dI-dC). Free probe and protein-bound probe were separated on a 5% polyacrylamide gel containing 2.5% glycerol and 0.5x TBE [50 mM Tris, 50 mM boric acid, and 50 mM EDTA (pH 8)]. The gel was dried and exposed to x-ray film at -80 C for 1–2 d. For supershift studies, the cell extract was preincubated with 0.1 µg Stat5a polyclonal antibody for 20 min at room temperature before the addition of labeled oligomer.

Statistical analysis
Data are given as mean ± SE. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni test. Values of P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Estradiol treatment inhibits the oPRL-induced increase in ntcp mRNA in OVX rats
Whereas treatment with oPRL increases Na+/TC cotransport and the expression of ntcp in OVX rats (7), treatment with high doses of ethynylestradiol decreases Na+/TC cotransport and expression of ntcp mRNA (43), even though estrogens also increase endogenous PRL secretion (44, 45). We therefore postulated that estrogens could also inhibit the PRL-induced increase in ntcp mRNA. To test this hypothesis, OVX rats were treated with oPRL (300 µg/d, 7 d) in the presence and absence of E2 treatment. Plasma levels of E2 were low in vehicle-treated rats (4.8 ± 1.4 pg/ml), consistent with ovariectomy, and were well within physiologic levels in E2-treated rats (SOL + E2, 40.1 ± 4.1 pg/ml; PRL + E2, 47.2 ± 2.7 pg/ml). Endogenous rat PRL levels were low (PRL + oil, 5.7 ± 0.3 ng/ml), consistent with the effectiveness of bromocriptine in blocking its secretion. Higher levels of rPRL in E2-treated rats (SOL + E2, 15.8 ± 2.2 ng/ml; PRL + E2, 14.7 ± 2.7 ng/ml) are consistent with the ability of estrogens to stimulate PRL secretion. Plasma levels of oPRL were comparable in PRL + oil (72.1 ± 24 ng/ml)- and PRL + E2 (79.2 ± 22 ng/ml)-treated rats. Ntcp mRNA expression was not different in OVX rats treated with oil or E2 (Fig. 2AGo). As demonstrated previously, oPRL treatment significantly increased ntcp mRNA expression (7). However, the oPRL-induced expression of ntcp mRNA in rats was significantly inhibited by the administration of E2 to the same levels as in rats treated with E2 alone (Fig. 2BGo).



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  		FIG. 2. The oPRL-induced expression of ntcp mRNA is inhibited by treatment with E2 in OVX rats. OVX rats were treated with bromocriptine to block endogenous PRL production. These rats were treated with oil or E2 (A), oPRL + oil, oPRL + E2, or Sol + E2 (B) for 7 d, as described in Materials and Methods. RNA was isolated from liver and subjected to Northern blot analysis using 32P-labeled rat ntcp cDNA probe. 28S rRNA was used as internal control to correct for the variance in RNA loading and transfer. Signals (top) were quantitated using the ImageQuant software and values shown in the bar graph (bottom). The values are expressed as mean ± SE. The numbers of rats used in each group are indicated in parentheses. *, P < 0.05 vs. PRL + E2 and Sol + E2.

 
Expression of ER{alpha} mRNA and protein is significantly increased in pregnant rats
Because many of the physiological actions of estrogens are mediated by ER, and ER expression can be modulated by multiple hormones (17, 27), we first examined the effects of pregnancy on the expression of ER{alpha} and ERß mRNA using real-time PCR. The results were normalized to the amount of ß-actin mRNA in each sample to minimize sample-to-sample variations that may have occurred during RNA extraction and quantification. Evolution of fluorescence vs. cycle number is shown in Fig. 3AGo. The relative amounts of ER{alpha} or ß-actin mRNA in each sample were calculated by using standard curves established from a series of successive dilutions of cDNA from normal rat ovary/oviduct (ER{alpha}) or liver (ß-actin) (data not shown). The methodology for real-time PCR, including the validation and calculation of data, was as described (42). ER{alpha} mRNA expression was significantly increased 2.6-fold in pregnancy relative to random cycling female controls (Fig. 3BGo). However, hepatic ERß mRNA was not detectable in either controls or pregnant rats (data not shown), as observed by other investigators (26). Expression of ER{alpha} protein was increased 2.2-fold in late pregnancy (Fig. 3Go, C and D), consistent with the 2.6-fold increase in its mRNA.



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  		FIG. 3. ER{alpha} mRNA and protein expression is increased in rat liver in pregnancy. A, Evolution of fluorescence during the amplification of ER{alpha} (upper) and ß-actin (lower), which was used as an internal control to normalize the transcripts of ER{alpha} in different samples. Total liver RNA (2 µg) isolated from control or rats 20–21 d pregnant was reverse transcribed to cDNA and the relative amounts of mRNA coding for ER{alpha} determined by real-time PCR, as described in Materials and Methods. B, ER{alpha} mRNA was quantified by analyzing data shown in A using a simultaneously produced standard curve and normalized to the amount of ß-actin present in the same sample. C, Western blot analysis of expression of ER{alpha} in total liver homogenate from control rats and rats 20–21 d pregnant. D, Quantitative analysis of ER{alpha} protein signals in C. The densitometric values were normalized to the mean value of ER{alpha} in control rats. The values are expressed as mean ± SE. The numbers of rats used in each group are indicated in parentheses. *, P < 0.05 vs. control.

 
Cotransfection of ER{alpha} or ERß suppresses PRL-induced, Stat5-mediated transactivation of ntcp in HepG2 cells
The in vivo data demonstrated that E2 antagonized the inductive effects of PRL on Ntcp expression, implying a cross-talk between PRL and estrogen signaling. To further examine the molecular basis of the interaction between PRL and estrogen signaling pathways, we used a transient cotransfection assay in HepG2 cells, in which we have shown that PRL and mouse PL transactivate ntcp promoter expression by activating the PRLRL-Stat5a signal transduction pathway (8, 9). HepG2 cells were transiently cotransfected with expression vectors for PRLRL, Stat5a, and ER{alpha}, ERß or empty vector pCMV5 together with the luciferase reporter construct containing the ntcp minimal promoter (4x 0.2pGL3) or the native ntcp promoter (p1237Luc) and treated with oPRL and E2. As reported previously, PRL activated the ntcp reporter construct 4x 0.2pGL3 (solid line), whereas addition of E2 had no effect on ntcp transactivation in cells cotransfected with the empty vector for ER (Fig. 4AGo). Cotransfection of ER{alpha} repressed PRL-induced ntcp reporter gene expression, and addition of E2 further repressed transactivation in a dose-dependent manner (Fig. 4BGo). Cotransfection of ERß also inhibited the inductive effect of PRL, although to a much lesser extent, so that inhibition was evident only at the highest concentration of E2 (Fig. 4CGo). This same experiment was repeated using the native ntcp promoter (p-1237Luc) to determine whether a similar effect was observed with the {gamma}-interferon activated sequences in their native context. As shown in Fig. 5Go, E2 treatment dose-dependently inhibited the PRL-induced transactivation of p-1237Luc in HepG2 cells cotransfected with ER{alpha} (Fig. 5BGo), and to a much lesser extent with ERß (Fig. 5CGo), but not with empty vector (Fig. 5AGo). Western analysis of Stat5a and PRLRL revealed that cotransfection of ER{alpha} did not affect their expression (Fig. 4DGo).



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  		FIG. 4. PRL-induced, Stat5a-mediated induction of 4x 0.2pGL3 transactivation is repressed by E2 and its receptors. HepG2 cells were transiently cotransfected with expression vectors for PRLRL (1 µg), Stat5a, empty vector pCMV5 (A), ER{alpha} (B), or ERß (C) together with the luciferase reporter construct containing the ntcp minimal promoter (4x 0.2pGL3), each 5 µg and treated with different concentrations of oPRL and E2, as indicated. A Microlumat LB 96P luminometer measured the activity of the luciferase reporter construct; ß-galactosidase was included for normalization of data. The oPRL-dependent fold induction was calculated relative to normalized luciferase activity in the absence of the hormone. Data represent mean ± SE of three independent experiments. In A, B, and C, the embedded bar graph represents the data obtained from cells treated with 1.0 µg/ml oPRL and more specifically shows the inhibitory effects of E2 in the presence of ERs. *, P < 0.05 vs. medium. D, Western analysis demonstrating that expression of transfected Stat5a and PRLRL (100 µg prg protein) was not affected by cotransfection of ER{alpha}.

 


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  		FIG. 5. E2 represses the oPRL-induced, Stat5a-mediated induction of native ntcp promoter transactivation through ERs. HepG2 cells were transiently transfected with expression vectors for PRLRL (1 µg), Stat5a empty vector pCMV5 (A), ER{alpha} (B), or ERß (C) together with the luciferase reporter construct containing the ntcp native promoter p-1237Luc, each 5 µg, and treated with different concentrations of oPRL and E2, as indicated. Luciferase activity analysis and data calculation were performed as described in legend of Fig. 4Go. *, P < 0.05 vs. medium.

 
We monitored the activity of ER{alpha} and ERß by introducing an estrogen-responsive indicator in which four copies of the ERE were linked to a thymidine kinase promoter of a luciferase expression plasmid (4x ERE-tk-luc). As shown in Fig. 6AGo, E2 treatment activated 4x ERE-tk-luc in a dose-dependent manner in HepG2 cells transfected with ER{alpha} or ERß but not empty vector. These data demonstrated that both ERs were able to transduce the E2 signal in HepG2 cells. Because ERß could not be detected in liver of control or pregnant rats, further studies focused on characterizing the inhibitory effects of ER{alpha} by cotransfecting increasing amounts of ER{alpha} expression plasmid. HepG2 cells were transiently cotransfected with expression vectors for PRLRL, Stat5a, increasing amounts of ER{alpha} as indicated, together with the ntcp minimal promoter reporter construct (4x 0.2pGL3), and treated with oPRL (1 µg/ml) in the presence or absence of E2 (10 nM). ER{alpha} inhibited transactivation of 4x 0.2pGL3 by PRL in a dose-dependent manner, whereas addition of E2 further enhanced this suppression (Fig. 6BGo).



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  		FIG. 6. A, E2 activated estrogen-responsive indicator gene 4x ERE-tk-luc. HepG2 cells were transfected with expression plasmid vectors for ER{alpha}, ERß, or empty vector together with an estrogen-responsive indicator gene 4x ERE-tk-luc, each 5 µg, and treated with different concentrations of E2. B, ER{alpha} repressed PRL-induced, Stat5a-mediated induction of 4x 0.2pGL3 transactivation in a dose-dependent manner. HepG2 cells were transfected with expression vectors for PRLRL, Stat5a, increasing amounts of ER{alpha} as indicated together with the ntcp minimal promoter reporter construct 4x 0.2pGL3 and treated with oPRL (1 µg/ml) in the presence or absence of E2 (10 nM). Luciferase activity analysis and data calculation were performed as described in legend of Fig. 4Go. *, P < 0.05 vs. medium.

 
ICI 182,780 reverses the inhibitory effect of E2 on ntcp transactivation by PRL
We next examined the effect of ICI 182,780, which is a pure ER{alpha} antagonist, on E2 inhibition of PRL-induced ntcp activation. HepG2 cells were cotransfected with expression vectors for PRLRL, Stat5a, and ER{alpha} or empty vector pCMV5, together with the ntcp minimal promoter reporter 4x 0.2pGL3 and treated with E2 and/or ICI 182,780 in the presence of PRL. ICI 182,780 alone showed no effect on PRL-induced 4x 0.2pGL3 activation in HepG2 cells cotransfected with ER{alpha} (Fig. 7Go, left panel). However, ICI 182,780 treatment completely reversed the E2-induced repression to that seen with ICI 182,780 alone or vehicle control (Fig. 7Go, left panel). When the empty vector of ER{alpha} was cotransfected, neither E2 nor ICI 182,780 had any effect on activation of 4x 0.2-pGL3 (Fig. 7Go, right panel). These results further indicate that the inhibitory effect of E2 on 4x 0.2pGL3 activation by PRL is ER{alpha} dependent.



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  		FIG. 7. ICI 182,780 reverses the inhibitory effect of E2 on PRL-induced, Stat5a-mediated induction of 4x 0.2pGL3 transactivation. HepG2 cells were transfected with ER{alpha} expression construct (left panel) or empty vector (right panel), each 5 µg, together with expression vectors for PRLRL (1 µg), Stat5a (5 µg), and the ntcp minimal promoter reporter construct 4x 0.2pGL3 (5 µg). Cells were treated with oPRL (1 µg/ml) in the presence or absence of E2 and/or ICI 182,780, as indicated. The results are presented as fold induction of luciferase activity normalized by ß-galactosidase in the absence of oPRL. Data represent mean ± SE of three independent experiments. *, P < 0.05, **, P < 0.01 vs. transactivation in the absence of E2.

 
P300 enhances PRL-induced 4x 0.2pGL3 transactivation but does not reverse ER{alpha}-mediated inhibition
The p300/CBP proteins represent a family of transcriptional coactivators that potentiate the activity of several groups of transcription factors, including ER{alpha} and Stat5 (46). Partitioning of limited amounts of shared transcriptional coactivators could explain the interference between transcriptional factors (47). Thus, if ER{alpha} and Stat5a were to share common coactivators, then partitioning of these coactivators to ER{alpha} could result in repression of Stat5a activity. Conversely, the supplementation of coactivators could reverse the inhibition of Stat5a activity by ligand-bound ER{alpha}. To test this possibility, a vector expressing the coactivator p300 was introduced in the cotransfection experiment. HepG2 cells were cotransfected with expression vectors for Stat5a, PRLRL, 4x 0.2pGL3, ER{alpha}, and p300 and then treated with oPRL and E2, (Fig 8Go). In the absence of ER{alpha} and E2, cotransfection of p300 enhanced PRL-induced, Stat5a-mediated 4x 0.2pGL3 transactivation (Fig. 8AGo). In the presence of ER{alpha} and E2, however, p300 did not reverse the E2-mediated repression of PRL-induced 4x 0.2pGL3 transactivation (Fig. 8BGo). Again, the presence of ER{alpha} did not affect the expression of p300 in HepG2 cells (Fig. 8CGo).



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  		FIG. 8. p300 enhances oPRL-induced, Stat5a-mediated 4x 0.2pGL3 transactivation (A) but does not reverse the inhibitory effects of ligand-activated ER{alpha} (B). A, HepG2 cells were transfected with expression vectors for PRLRL (1 µg), Stat5a (5 µg), the ntcp minimal promoter reporter construct 4x 0.2pGL3 (5 µg) together with expression vectors encoding p300 or its empty vector (5 µg). Cells were treated with oPRL as indicated for 40 h. Luciferase activity analysis and data calculation were performed as described in legend of Fig. 4Go. *, P < 0.05 vs. vector. B, HepG2 cells were transfected with expression vectors for ER{alpha} (5 µg), PRLRL (1 µg), Stat5a (5 µg), 4x 0.2pGL3 (5 µg), and vector coding for p300 (right) or its empty vector (left), each 5 µg. Cells were treated with 0.1 µg/ml oPRL and increasing concentration of E2 as indicated. C, Western analysis demonstrating that expression of transfected p300 was not affected by cotransfection of ER{alpha}. Luciferase activity analysis and data calculation were as described in legend of Fig. 4Go. *, P < 0.05, **, P < 0.01 vs. transactivation in the absence of E2.

 
Ligand-activated ER{alpha} decreased the tyrosine phosphorylation and the DNA-binding activity of Stat5a
To further characterize the inhibitory effect of ER{alpha} on the PRL signal transduction pathway, we determined ER{alpha} effects on Stat5a phosphorylation and DNA binding activity. For this purpose, we performed Western blot analysis and EMSA of whole-cell extracts from HepG2 cells cotransfected with expression plasmids for PRLRL, Stat5a, and ER{alpha} or its empty vector. Five hours after transfection, cells were incubated with fresh medium for a further 36 h and then treated with 1 µg/ml oPRL and 10 nM E2 for 1 h and whole-cell lysate prepared for Western analysis and DNA binding activity. Whereas cotransfection of ER{alpha} did not affect the expression levels of Stat5a protein (Fig. 9AGo), cotransfection of ER{alpha} resulted in decreased tyrosine phosphorylation of Stat5a in the presence of E2 and PRL, compared with empty vector (Fig. 9BGo, lanes 3 and 4 vs. lanes 1 and 2). Omission of Stat5a in the cotransfection was used to identify Stat5a in the Western blot (Fig. 9Go, A and B, last lane). In parallel with the decreased activation of Stat5a by tyrosine phosphorylation, the presence of ER{alpha} and E2 decreased DNA binding activity of Stat5a to the ß-casein Stat5 response element (Fig. 9CGo, lanes 3 and 4 vs. lanes 1 and 2) as well as the ntcp Stat5 response element (Fig. 9DGo, lanes 3 and 4 vs. lanes 1 and 2). The DNA-protein complexes were supershifted in the presence of Stat5a antibody (Fig. 9Go, C and D, last lane), and no complex was detected in the absence of Stat5a cotransfection, confirming the specificity of Stat5a-DNA complexes (data not shown).



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  		FIG. 9. E2 decreases tyrosine phosphorylation and the DNA-binding activity of Stat5a in the presence of ER{alpha}. HepG2 cells were transfected with expression vectors for ER{alpha} (lanes 3 and 4) or its empty vector pCMV5 (lanes 1 and 2), each 5 µg, PRLRL (1 µg), and Stat5a (5 µg). The transfected cells were exposed to 1 µg/ml oPRL and 10 nM E2 for 1 h, and whole-cell extracts were prepared and 80 µg protein subjected to Western analysis with anti-Stat5a antibody (A) or anti-phosphor-Stat5a antibody (B) and EMSA using 32P-labeled consensus oligonucleotide from the bovine ß-casein promoter (C) or the ntcp promoter corresponding to -918 to -898 bp (D). In the Western blot, omission of transfection of Stat5a confirmed the specificity of Stat5a detection (last lane). In EMSA, the supershift assays were performed by using anti-Stat5a antibody to confirm the presence of Stat5a in the protein-DNA complex (last lane).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study characterized the effects of physiological levels of E2 on the ability of PRL to increase expression of ntcp, the key sodium-dependent bile salt transporter that mediates the uptake of bile salts from blood across the basolateral plasma membrane into the hepatocyte. In vivo studies demonstrated that, whereas PRL increased ntcp mRNA expression in OVX rats, ntcp expression was not increased in OVX rats treated with both PRL and E2. Furthermore, studies in HepG2 cells cotransfected with the PRLRL, Stat5a, and ER{alpha} demonstrated that in the presence of ER{alpha}, E2 markedly repressed the PRL-induced transactivation of both the native ntcp promoter (p1237Luc) and a Stat5a reporter construct (4x 0.2pGL3) containing the ntcp minimal promoter. The inhibitory effects of E2 were mediated, at least in part, by inhibiting tyrosine phosphorylation and binding of Stat5a to Stat5 response elements in the ß-casein and ntcp promoters. The inhibitory actions of E2 were dependent on the presence of ER{alpha} and were antagonized by the pure ER antagonist, ICI 182,780. These data in HepG2 cells provide a mechanistic basis for E2 inhibition of PRL-mediated increases in ntcp expression in vivo. These data also provide a likely mechanism for the observation that in pregnancy, when circulating PL levels are high and ER{alpha} expression is increased, hepatic ntcp expression does not increase. We have previously demonstrated that PL increases ntcp expression in primary cultures of hepatocytes and transactivates the ntcp promoter using the same signal transduction pathway as PRL, i.e. via the PRLRL-Stat5a pathway (8). E2 and ER{alpha} also inhibited PL-induced ntcp promoter transactivation in HepG2 cells transiently transfected with Stat5a and PRLRL (data not shown). The combination of increased plasma E2 levels and increased expression of hepatic ER{alpha} in late pregnancy thus likely prevents the increased expression of ntcp anticipated by the actions of PL alone.

It is generally assumed that transactivators stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex and increasing the transcription initiation rate of RNA polymerase II (24). Efficient transcription requires additional positively acting factors, termed coactivators, which act as a bridge between nuclear receptors and the transcriptional machinery. The p300/cAMP response element-binding protein (CREB)-binding protein (CBP) proteins represent a family of transcriptional coactivators that potentiate the activity of several groups of transcriptional factors, including cAMP response element-binding protein (48, 49, 50), activator protein-1, nuclear receptors (including ERs) (51, 52), and, importantly, the Stat proteins (53, 54). Therefore, the relative ability of one transcriptional factor vs. another to recruit a limiting amount of p300/CBP into stable preinitiation complexes could explain the negative cross-talk between these two transcription factors. We first postulated that ER{alpha} exerted its inhibitory effects by competing with Stat5a for a limiting pool of p300/CBP. Although the overexpression of p300 enhanced the PRL-induced Stat5-mediated transcriptional activation (Fig. 8Go), as observed by others (46), it was not able to overcome the inhibitory effects of ER{alpha}, indicating involvement of other mechanisms.

A number of studies have demonstrated an interaction between the Stat5 and nuclear receptor signal transduction pathways. PRL and hydrocortisone have been shown to act synergistically to enhance transcription of ß-casein in mammary epithelial cells (55). Protein-protein interactions between the glucocorticoid receptor and Stat5 were subsequently shown to mediate this synergy (32) and could be attributed to increased Stat5 tyrosine phosphorylation and enhanced DNA-binding activity of Stat5 in the presence of the glucocorticoid receptor (56). In contrast, cotransfection of ER in COS cells diminished Stat5-mediated transcription from the ß-casein promoter (32). Subsequent studies demonstrated that ER decreased tyrosine Stat5 phosphorylation and DNA binding, both in the presence and absence of E2 and that the decrease in tyrosine phosphorylated Stat5 was not due to increased rates of its dephosphorylation (56). Furthermore, detailed studies showed direct physical interactions between the ER DNA-binding domain and Stat5 (35). In these studies, however, binding of E2 to the ER ligand-binding domain was required for ER{alpha}-mediated inhibition of PRL-induced Stat5 activation. In further contrast to the present results, Faulds et al. (35) found that ER{alpha} and ERß were equally effective in inhibiting PRL-induced Stat5 transcriptional activity of the ß-casein promoter. These differences could be due to differences in the ß-casein vs. ntcp promoter or between COS and HepG2 cells. In vivo studies have shown that Stat5 is localized to the nucleus in both female and male ER{alpha}-deficient mice, whereas in wild-type mice, nuclear localization of Stat5 is observed only in male mice (57). The presence of nuclear Stat5 in ER{alpha}-deficient female mice led to repression of the female-specific Cyp2a4 and expression of the male-specific Cyp2d9 genes (57). Thus, in wild-type mice, ER{alpha} inhibits the nuclear localization of Stat5 in livers of female mice, leading to feminization of expression of cytochrome P450.

In agreement with these earlier studies, the present studies provide strong evidence that in pregnancy, E2 and ER{alpha} inhibit the activation of Stat5a by attenuating tyrosine phosphorylation, which is critical for its nuclear localization, thus blocking PL-mediated signal transduction and activation of ntcp. It is noticeable that the expression of ER{alpha} also exerted inhibitory effects on the PRL-induced transactivation of both the native ntcp promoter (p1237Luc) and a Stat5a reporter construct (4x 0.2pGL3) containing the ntcp minimal promoter (Figs. 4–6GoGoGo). A similar phenomenon was observed by other investigators (56). Although the detailed mechanisms are not known, the expression of other cotransfected genes (Stat5a, PRLRL, and p300) was not affected by ER{alpha} (Figs. 4DGo and 8CGo).

It is also possible that direct binding of ER{alpha} to the ntcp promoter contributes to its inhibitory effects on ntcp transactivation. Three half-EREs (GGTCA) are located at positions -780 to -776, -182 to -176, and -84 to -81 bp upstream of the transcriptional start site of ntcp; the latter half-ERE is present in the minimal promoter of ntcp. ERE half-sites, bound by ERs, have been shown to participate in the regulation of many genes (58, 59). It will be of interest to determine whether activated ER{alpha} is able to bind these half-EREs and thereby inhibit the initiation of ntcp transcription by Stat5. Furthermore, studies using mutation analysis of these ERE half-sites should prove insightful in this regard.

In summary, the present study showed that E2 blocked the PRL-mediated increase in ntcp mRNA expression in rats. In HepG2 cells, E2 potently repressed PRL-induced Stat5 transcriptional activity of ntcp promoter constructs in the presence of ER{alpha}; ERß had much lesser effects. Furthermore, the pure ER antagonist ICI 182,780 reversed the E2-dependent inhibition of PRL-induced transactivation of ntcp. Finally, E2-activated ER{alpha} decreased the tyrosine phosphorylation and DNA-binding activity of Stat5a, indicating that the inhibitory effect of E2 was mediated at least in part by interfering with PRL receptor-mediated signal transduction. These data demonstrate the physiological significance of cross-talk between ER{alpha} and Stat5a in liver, in which both proteins are expressed. These data also establish a novel mechanism by which expression of ntcp, an important bile acid transporter in liver, can be regulated by multiple hormones. These findings strongly support the hypothesis that the increased expression of hepatic ER{alpha} and elevated circulating estrogen levels inhibit the ability of PL to increase ntcp expression in pregnancy.


   Footnotes
 
This work was supported by Public Health Service Grants DK46923 (to M.V.), HD36879 (to O.-K.P.-S.), and Training Grant ES07266 (to M.W.).

Abbreviations: CBP, cAMP response element-binding protein (CREB) binding protein; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; ntcp, Na+/taurocholate cotransporting polypeptide; OVX, ovariectomized; PL, placental lactogen; PRL, prolactin; PRLRL, long form of the PRL receptor; Stat, signal transducers and activators of transcription.

Received June 13, 2003.

Accepted for publication December 9, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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