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Endocrinology Vol. 139, No. 11 4581-4589
Copyright © 1998 by The Endocrine Society

ARTICLES

Orphan Receptor Hepatocyte Nuclear Factor-4 Antagonizes Estrogen Receptor {alpha}-Mediated Induction of Human Coagulation Factor XII Gene1

A. Farsetti, F. Moretti, M. Narducci, S. Misiti, S. Nanni, M. Andreoli, A. Sacchi and A. Pontecorvi

Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute (A.F., F.M., M.N., S.M., S.N., A.S., A.P.); the Institute of Experimental Medicine, National Research Council/Second Chair of Endocrinology, University of Rome La Sapienza (A.F., F.M., S.M., M.A.); and the Institute of Medical Pathology, Catholic University (A.P.), Rome, Italy

Address all correspondence and requests for reprints to: Alfredo Pontecorvi, M.D., Catholic University and Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, 00158 Rome, Italy.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Factor XII (FXII) is a liver-specific zymogen involved in the regulation of hemostasis, particularly in the activation of fibrinolysis. Transcription of the FXII gene is stimulated by estrogens through specific interaction of the estrogen receptor {alpha} (ER{alpha}) with an estrogen response element present on FXII promoter. Interestingly, the magnitude of ER{alpha} induction in liver HepG2 cells is much lower than in NIH3T3 fibroblasts, suggesting that cell-specific factors may modulate ER{alpha}-dependent trans-activation. Comparative footprinting analysis of FXII promoter (from nucleotides -181 to +49) in liver vs. non-liver cell environments allowed identification of four deoxyribonuclease I-protected sites only in the presence of HepG2 nuclear extracts. Computerized homology search identified sites III and IV as consensus binding sequences for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4), formerly an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors. In transient transfection assays in NIH3T3 cells, HNF-4 significantly inhibited (70%) estrogen induction of FXII promoter while not affecting basal promoter activity. Conversely, HNF-4 did not inhibit estrogen inducibility of FXII promoter in HepG2 cells due to the high endogenous levels of HNF-4 protein. In gel shift assays, HNF-4, either present in HepG2 nuclear extracts or generated by in vitro transcription/translation, specifically bound FXII promoter. This interaction is strictly required in eliciting the antagonistic effect because in NIH3T3 cells, selective mutations of sites III and IV abrogated HNF-4 inhibitory properties. In the liver-specific environment, the same mutant construct exhibited higher estrogen-dependent inducibility compared with native promoter. Rescue of estrogen responsiveness was also achieved using a dominant negative HNF-4, which counteracted endogenous HNF-4 activity. In conclusion, our findings address a direct role for HNF-4 in modulating estrogen-dependent transcription of the FXII gene promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FACTOR XII (FXII) is a liver-specific zymogen acting at the cross-point between the coagulation and the fibrinolytic enzymatic cascades (1). Upon binding to collagen or to negatively charged surfaces, usually exposed after tissue injury and inflammation or present on artificial membranes and surgical prosthesis, FXII becomes highly susceptible to limited proteolysis by kallikrein (2). This enzymatic cleavage converts FXII into a two-chain disulfide-linked active enzyme (FXIIa), which exhibits serine protease-like activity at its carboxyl-terminus and is capable of activating the fibrinolytic system as well as initiating blood coagulation (3).

The primary physiologic role of FXII in activating the intrinsic pathway of blood coagulation has recently been challenged by the following observations: 1) the FXII molecular structure more closely resembles that of fibrinolytic proteins, particularly the tissue plasminogen activator, than that of other coagulation factors (1); 2) FXI, the natural FXII substrate along the intrinsic blood coagulation pathway, may be activated independently from FXII (4); 3) FXII is able to activate the potent vasodilating agent bradykinin, which causes dilation of vessels occluded by thromboembolic processes and further stimulates fibrinolysis by inducing the release of tissue plasminogen activator from the vascular endothelium (5); and 4) a major role of FXII along the fibrinolytic rather than the coagulation pathway is finally supported by the observation that, contrary to congenital defects of other coagulation factors, which result in mild to severe bleeding disorders, inherited deficiency of FXII is usually complicated by thromboembolic episodes (6).

Constituents of the enzymatic coagulation and fibrinolytic cascades require a very tight regulation to ensure constant plasma concentrations and consistent levels of activation, as derangement in their activity may be highly deleterious to the entire organism. Hormonal regulation of several components of these enzymatic cascades has been thoroughly demonstrated (7, 8, 9, 10). In particular, clinical studies indicated that FXII is extremely sensitive to estrogen stimulation; its titer is specifically increased after low dose synthetic estrogen administration (5 µg ethinyl estradiol), a dosage that exhibits a biologic potency similar to that elicited by natural estrogenic compounds commonly used in postmenopausal estrogen replacement therapy (11). The selective induction of FXII plasma levels by low dose estrogens may, therefore, represents an additional mechanism for the overall reduction in cardiovascular disease morbidity and mortality associated with estrogen replacement therapy in postmenopausal women (12).

We previously demonstrated that FXII gene expression, both in vivo and in vitro, is specifically regulated by estrogens at the level of transcription through the interaction of liganded estrogen receptor {alpha} (ER{alpha}) with a specific estrogen response element (ERE), identified in the context of FXII promoter (8). Interestingly, estrogen induction of FXII gene transcription was significantly lower in the physiological environment of human hepatoma cells than in mouse fibroblasts, suggesting that cell-specific factors may modulate FXII estrogen responsiveness. In the present study we attempted to identify putative cell-type specific proteins that may modify ER trans-activation properties. In particular, we identified the orphan receptor hepatocyte nuclear factor-4 (HNF-4) as a potent repressor of ER-mediated transcriptional regulation of the FXII gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection
Mouse fibroblast NIH3T3 and human hepatoma HepG2 cells were cultured in DMEM containing 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Two days before transfection, cells were seeded at a density of 1.2 x 106/175 cm2 flask in phenol red-free DMEM containing 10% hormone-stripped FCS (13). Plasmid transfections, chloramphenicol acetyltransferase (CAT) assays, and quantitation of CAT activity were performed as previously described (14). Briefly, 5 µg reporter plasmid were electroporated into HepG2 or NIH3T3 cells with or without expression plasmid, for either ER{alpha} (5 µg) or HNF-4 (5 µg) or for 5 µg both expression plasmids. Empty vector was added to maintain 15 µg expression plasmid/60-mm petri dish transfection. Transfections also included 250 ng cytomegalovirus (CMV)-ß-galactosidase (ß-gal) for monitoring transfection efficiency. After incubation for 72 h in the presence or the absence of 10-7 M 17ß-estradiol (E2), cells were harvested, resuspended in 150 µl 0.25 M Tris-HCl, pH 8, and lysed with three freeze-thaw cycles. Twenty microliters of the lysate were assayed for ß-gal activity, calculated as follows: units = (absorbance at 420 nm x 380)/min, where 380 is a constant such that 1 U is equivalent to 1 nmol of the substrate O-nitrophenol ß-D-gal hydrolyzed/min (15). CAT activity was determined by a phase extraction method (14) in 50 µl cell extract lysate. When needed, CAT assay was repeated using dilutions to assure the linearity of the assay (3,000–120,000 cpm/reaction). The background, corresponding to extract from cells transfected with pUC18 alone, was less than 1,000 cpm. After subtraction of background, CAT activity (in counts per minute) was normalized to the units of ß-galactosidase activity.

Electrophoretic mobility shift assay
Double-stranded oligonucleotides containing sequences of FXII (+7/+38) and {alpha}1-antitrypsin (-101/-128) promoters were assayed by native gel electrophoresis for binding to in vitro transcribed/translated HNF-4 (TNT-Coupled Wheat Germ Extract System, Promega Corp., Madison, WI). In vitro binding reactions were performed in a final volume of 20 µl in the presence of 10 mM Tris-HCl (pH 7.5), 30 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5 mM MgCl2, 10% (vol/vol) glycerol, and 1 mg poly(dI-dC) (Midland Certified Reagent, Midland, TX). Gel shift with HepG2 nuclear extract were performed using DNA binding conditions previously described (16). Samples were loaded onto a 6% polyacrylamide gel and electrophoresed for 3 h at 150 V using 0.5 x TBE (45 mM Tris borate, 45 mM boric acid, and 2 mM EDTA) as running buffer. To some samples anti-HNF-4 polyclonal antibody was added after incubation with the 32P-labeled probe. For competition experiments, increasing concentrations of unlabeled oligonucleotide containing FXII-HNF-4-binding site were added to the binding mixture for 20 min before addition of the 32P-labeled probe. Oligonucleotides used in the analysis of HNF-4 binding were 5'-ATCTGGACTCCTGGATAGGCAGCTGGACCAAC-3' for FXII and 5'-CAGCCAGTGGACTTAGCCCCTGTTTGCT- 3' for {alpha}1-antitrypsin.

Deoxyribonuclease I (DNase) footprinting
Nuclear extracts from HepG2 and NIH3T3 cells were prepared according procedures described by Naeve et al. (17). The plasmid PT-CAT181 was linearized with BamHI, dephosphorylated with calf intestinal alkaline phosphatase, and labeled at the 5'-end (the upper strand) with [{gamma}-32P]ATP using T4 polynucleotide kinase. The FXII promoter fragment was released by PstI digestion and purified by PAGE. DNase I footprinting was performed according to the method of Jones et al. (18). Briefly, the labeled fragment (25 x 103 cpm/reaction) was incubated in 50 ml binding mixture either with or without variable amounts of HepG2 or NIH3T3 nuclear extract for 30 min at room temperature. Then 5 ml Ca2+/Mg2+ solution (10 mM MgCl2 and 4 mM CaCl2) were added followed by the addition of 3 U/ml DNase I (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). One minute later, the digestion was stopped by the addition of 140 ml stop solution containing 768 mM sodium acetate, 128 mM EDTA, 0.56% SDS, and 256 mg/ml yeast RNA. The mixture was precipitated with ethanol, washed with 70% ethanol, and subjected to electrophoresis through an 8% polyacrylamide sequencing gel containing 7 M urea.

Western blot analysis
NIH3T3 or HepG2 cells (1.5 x 105) were lysed directly in 15 µl protein sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 1.1% bromophenol blue]. The extracts were subjected to ultrasound to reduce viscosity and were boiled for 5 min before electrophoresis on a 12% polyacrylamide gel containing SDS. Proteins were transferred to a nitrocellulose membrane by electroblotting. After blocking of nonspecific protein-binding sites for 1 h at room temperature in TBST [10 mM Tris-HCl (pH 7.8), 150 mM NaCl, and 0.05% (vol/vol) Tween-20] containing 5% (wt/vol) nonfat dry milk, the blots were incubated with anti-HNF-4 (19) or anti-ER polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:1000 dilution for 1 h at room temperature. After three washes of 5 min each in TBST, the secondary peroxidase-conjugated antirabbit antibody (Santa Cruz Biotechnology) was added at a 1:2000 dilution and left for 45 min at room temperature. Blots were washed again in 10 mM Tris-HCl (pH 8) and 150 mM NaCl, and peroxidase activity was detected by autoradiography using an enhanced chemiluminescence system (ECL, Amersham, Arlington Heights, IL).

Plasmids
Plasmid PT-CAT-181 has been previously described (8). PT-CAT181 mut, in which both HNF-4-binding sites were mutated, was generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The following oligonucleotide and its complementary sequence were used as primers (mutated nucleotides are underlined): 5'-CCTATTGATCAAAAAACCTGGATAGGCAGCAAAAAAAACGGACGG-3'.

PT-CAT{Delta}181, containing a deletion of the downstream HNF-4 binding motif, was generated by ligation of a PCR-generated 210-bp genomic fragment of FXII promoter, from nucleotides -181 to +27, into the PstI/SalI sites of the PT-CAT expression vector (8). The following oligonucleotides were designed as primers: 5'-AATGGCGAGGATCCGTCGACATCTAGAAAAGAGAGGAG-3' and 5'-AATCTAGAGCA-TGCCTGCAGGCCTATCCAGGAGTCCAG-3'.

The expression vector VIT-CAT, containing the Xenopus laevis vitellogenin B1 gene promoter (nucleotides -596/+8) fused to the CAT reporter gene (19), was a gift from Dr. J. Tata (Medical Research Council, London, UK). pSG5-HEO was a gift from Prof. P. Chambon (Institut de Génétique et de Biologie Moléculaire e Cellulaire, Strasbourg, France). HNF-4 expression plasmid pLEN4S, containing the full-length rat HNF-4{alpha}1 complementary DNA under the control of the human metallothionein promoter and the simian virus 40 enhancer (20), was a gift from Dr. F. Sladek (University of California-San Francisco, Riverside, CA). In vitro translated HNF-4 was made using as template the expression vector pMT7-rHNF-4 (21). HNF-4 mutant expression construct containing deletion of DNA-binding domain, HNF-4DBD (22), was a gift from Dr. Gavin Kelsey from the Babraham Institute (Cambridge, UK). The expression plasmid CMV-ß-galactosidase, containing a fusion between the CMV long terminal repeat and the ß-gal gene (16), was used as an internal control for monitoring transfection efficiency.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of protein-binding sites in FXII gene promoter by DNase I footprinting
The lower magnitude of induction of FXII transcription by liganded ER in human hepatoma (HepG2) compared with NIH3T3 cells (8), suggests that cell-type specific factors may modulate ER{alpha} trans-activation properties. Attempts were made to identify and locate nuclear proteins which, by interacting with FXII promoter sequences, may be involved in the regulation of FXII gene transcription in a cell type-specific environment. DNase I footprint assays were therefore performed by incubating FXII promoter fragment (from nucleotides -181 to +49) with nuclear extracts derived from HepG2 and NIH3T3 cells. In the presence of nuclear extracts from HepG2, but not from NIH3T3, cells, four major binding sites could be defined within the FXII 5'-flanking region (Fig. 1AGo). All protected areas lay within a promoter fragment approximately 100 bp in length that spanned the transcription start site (+1). Site I encompassed the ERE, previously identified at position -44/-31 and found to mediate functional interaction with liganded ER{alpha} (8). As expected, this site was weakly protected by DNase I digestion because HepG2 cells contain low ER levels (23). Site II coincided with the major transcription start site of the FXII promoter (24). Sites III and IV represented two well defined protected regions, the former being also characterized by DNase I hypersensitivity. Computerized homology search of published signal sequences identified protected regions III and IV as putative binding sites for HNF-4, a liver-enriched transcription factor until recently classified as an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors (20, 25). Interestingly, HNF-4 consensus sequences were both positioned downstream from the transcription start site (+1) and, in agreement with Ramji et al. (26), apparently arranged as a potential bipartite motif (Fig. 1BGo).



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  		Figure 1. FXII promoter contains multiple binding sites for hepatic cell nuclear proteins. A, DNase I footprint analysis showing FXII proximal promoter in the absence (w/o NE) and presence of increasing amounts (2.5, 5, 10, and 20 µg) of NIH3T3 (NIH3T3 NE) or HepG2 (HepG2 NE) cell nuclear extracts (NE). A probe spanning nucleotides -181 to +49 in the FXII gene was end labeled at -181, then digested with DNase I. Maxam-Gilbert G+A sequencing of the same fragment was also performed (G + A). The protected regions (I–IV) are shown on the right beside the autoradiograms. Site I corresponds to the previously identified ERE; site II coincides with the major transcription start site (+1); sites III and IV are homologous to the consensus sequences of the liver-enriched transcription factor HNF-4. The figure depicts a representative experiment of three independent experiments, each performed with newly prepared nuclear extracts. B, Sequences and positions of protected motifs in the FXII promoter. C, Western blot analysis of ER and HNF-4 endogenous proteins from NIH3T3 and HepG2 cells. The higher mol wt signal detected in NIH3T3 cells after incubation with anti-ER antibody (upper panel) represents a nonspecific band.

 
To validate results from DNase I footprinting assay, Western blot analysis of whole cell extracts from HepG2 and NIH3T3 cells was performed (Fig. 1CGo). Anti-ER and anti-HNF-4 antibodies specifically reacted with two single protein bands, corresponding to the expected protein products of 66 and 54 kDa, respectively, only in the context of the liver-derived cell line. These results show that HNF-4 is a fairly abundant protein in liver cells, in agreement with previous reports (27). The human hepatoma HepG2 cell line (28) has been shown to retain many of the differentiated characteristics of normal hepatocytes, including the expression of ER (23), although at levels not sufficient to trans-activate an ERE-containing reporter in the presence of ligand, as it is the case for ER-positive breast cancer MCF7 cells (29).

These results suggest that HepG2, but not NIH3T3, cells contain cell-specific transcription factors, the most likely being the orphan receptor HNF-4, that interact with FXII promoter and may contribute to modulate ER{alpha}-mediated induction of the FXII gene.

HNF-4 antagonizes ER{alpha}-mediated induction of FXII gene promoter activity
As functional interaction between members of the nuclear receptor superfamily is one of the distinctive features of their mode of action, we investigated whether coexpression of the orphan receptor HNF-4 with a traditional member of the superfamily, such as ER{alpha}, would affect either basal or ER{alpha}-dependent FXII transcriptional activation.

NIH3T3 and HepG2 cells, cultured in the absence or presence of E2, were transiently cotransfected with a reporter plasmid containing the first 230 bp of FXII promoter (PT-CAT181) and ER{alpha} and/or HNF-4 expression vectors. The results obtained in NIH3T3 cells confirmed our previous observation of a strong ER{alpha} induction of FXII promoter activity in the presence of E2, with an overall increase of about 30-fold (Fig. 2AGo). Transfection of HNF-4 alone did not modify the basal activity of the FXII minimal promoter (see Fig. 5Go). Conversely, when cotransfected with ER{alpha}, HNF-4 produced a significant inhibition (70%; P < 0.01) of estrogen-stimulated CAT activity, thus suggesting an interference with ER{alpha} trans-activation properties. Similar results were obtained using an expression vector for the recently cloned human HNF-4{alpha}1 (data not shown). No effects of the empty vector, used to drive HNF-4 expression, on FXII promoter inducibility by E2 were observed (see Figs. 5Go and 7Go).



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  		Figure 2. Exogenous expression of HNF- 4 differentially affects ER-dependent trans-activation of FXII promoter. NIH3T3 (A) or HepG2 (B) cells, cultured in the presence or absence of E2, were cotransfected by electroporation with ER{alpha} expression vector (5 µg), PT-CAT181 (5 µg) as reporter, CMV-ß-gal (250 ng) for transfection efficiency, and either HNF-4 or empty vector (5 µg). Cells were incubated for 72 h, harvested, and assayed for CAT and ß-gal activities. The fold E2 induction is calculated as the ratio of normalized CAT activity (counts per min/U ß-gal) in the presence or absence of hormone (± E2). Results represent the average (±SE) of eight independent experiments, each performed in duplicate. HNF-4 significantly (P < 0.01) repressed E2/ER{alpha} induction on PT-CAT181 only in NIH3T3 cells. No effects of the empty vector on estrogen inducibility of FXII promoter were observed.

 


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  		Figure 5. Site-directed mutagenesis of HNF-4-binding site abrogates HNF-4-antagonizing action in NIH3T3 cells. Wild-type (PT-CAT181; A), site-directed mutated (PT-CAT181 mut; B), and HNF-4-deleted (PT-CAT{Delta}181; C) FXII reporters (5 µg each) were cotransfected with ER{alpha}- and HNF-4-expressing vectors (1:1 ratio) into NIH3T3 cells as described in Fig. 2Go. Data are expressed as CAT activity (counts per min/units ß-gal x 103) in the absence (-E2) or presence (+E2) of E2. Results represent the average (±SE) of four independent experiments, each performed in duplicate.

 


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  		Figure 7. Dominant negative HNF-4 is able to rescue the estrogen inducibility of native FXII promoter by counteracting endogenous HNF-4. Native FXII promoter CAT reporter was cotransfected in HepG2 cells with expression vectors for ER{alpha}, HNF-4, and a DNA-binding domain-deleted HNF-4 (HNF-4DBD) or empty vector (-) using the same conditions as those described in Fig. 5Go. Data represent the average of four independent experiments, each performed in duplicate.

 
As previously noted, in liver-derived HepG2 cells, liganded ER{alpha}-mediated trans-activation of FXII promoter resulted in a lower magnitude of induction (~6-fold) compared with that in NIH3T3 cells (Fig. 2BGo). Interestingly, no inhibition of liganded ER{alpha}-dependent stimulation of FXII promoter activity was elicited after coexpression of HNF-4 protein. This lack of effect by HNF-4 could be due to the high endogenous levels of the factor constitutively expressed in the human hepatoma-derived HepG2 cell line (Fig. 1CGo) (27, 30, 31).

To investigate whether the orphan receptor HNF-4 is a general repressor of ER{alpha}-mediated transcriptional activity, parallel transfection assays were performed using a reporter plasmid containing the liver-specific vitellogenin B1 native promoter (VIT-CAT), characterized by the presence of a canonical ERE. As shown in Fig. 2Go, A and B, cotransfection of HNF-4 did not modify either basal or ER{alpha}-dependent stimulation of VIT-CAT activity in NIH3T3 or HepG2 cells.

These results indicate that the orphan receptor HNF-4, usually known as a liver-enriched transcription activator, is able to negatively modulate ER{alpha} trans-activation of a native liver-specific gene promoter. The different effects exerted on ER{alpha}-mediated induction of FXII promoter compared with that of vitellogenin, suggest that specific structural determinants, present in the context of the FXII promoter, are required for HNF-4 to elicit its repressive action.

Analysis of DNase I-protected regions III and IV on FXII gene promoter
To investigate whether DNase I-protected regions III and IV, which contain consensus sequences for binding to HNF-4, are able to interact with the liver-enriched orphan receptor, electrophoretic mobility shift assays were performed. Incubation of a 32P-labeled oligonucleotide, spanning the putative bipartite FXII HNF-4 motif, with HepG2 nuclear extracts, resulted in the formation of a retarded band (Fig. 3Go, lane 2) that was specifically supershifted (lane 3) by the addition of an anti-HNF-4 polyclonal antibody (gift from Dr. F. Sladek). These data indicate that the DNA-protein complex is likely to contain HNF-4, which is endogenously expressed by HepG2 cells. Increasing concentrations of unlabeled oligonucleotide (at 50- and 100-fold molar excesses) efficiently competed for binding of the endogenous HNF-4 protein (Fig. 3Go, lanes 4 and 5, respectively). A similar retarded band was observed after incubation of HepG2 nuclear extracts with a labeled oligonucleotide containing the HNF-4-binding site of the {alpha}1-antitrypsin gene (Fig. 3Go, lanes 6 and 7). This band was also supershifted (lane 8) following incubation with the anti-HNF-4 antibody. Upon addition of anti-HNF-4 antibody we observed the appearance of a faster electrophoretic migrating band with FXII probe alone, and a significant increase in the specific complex either with the FXII or the {alpha}1-antitrypsin oligonucleotides. Whether these phenomena may be accounted for by 1) a stabilization of the complex as we previously reported for ER{alpha} and FXII ERE in mobility shift assays (8), 2) a complex formed between FXII promoter and a putative monomeric HNF-4, or 3) technical artifacts remains to be elucidated.



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  		Figure 3. The FXII promoter HNF-4-binding site forms a specific complex with HepG2 nuclear extracts. A gel shift assay was performed with HepG2 nuclear extracts and the FXII or {alpha}1-antitrypsin HNF-4-binding site as probes (FXII and {alpha}1AT, respectively). Lanes 1 and 6, Probes alone; lanes 2–5 and 7–8, after incubation with HepG2 nuclear extracts and in the presence of 50- and 100-fold molar excesses (lanes 4 and 5) of unlabeled FXII HNF-4 probe; lanes 3 and 8, plus a specific antiserum raised against HNF-4. Open arrow, HNF-4/DNA-specific complex; filled arrow, supershift of the HNF-4/DNA complex by anti-HNF-4 antibody.

 
These results indicate that HNF-4, present in HepG2 nuclear extracts, is able to interact with a specific region of FXII promoter encompassing two putative HNF-4 binding motifs.

Binding of HNF-4 to putative HNF-4 consensus sequences of the FXII gene promoter
To further characterize putative HNF-4-binding sites, we tested the ability of HNF-4 protein, generated by in vitro transcription/translation, to directly interact with FXII promoter in electrophoretic mobility shift assays. Incubation of increasing concentrations of HNF-4 protein with a 32P-labeled oligonucleotide encompassing both putative HNF-4-binding sites of FXII promoter resulted in the formation of a retarded band (Fig. 4Go, lanes 3–5) that was efficiently competed out by the addition of 50-, 100-, and 250-fold unlabeled specific oligonucleotide (Fig. 4Go, lanes 6–8). A similar retarded complex was obtained after incubation of HNF-4 protein with an oligonucleotide spanning the well characterized {alpha}1-antitrypsin HNF-4 motif (Fig. 4Go, lane 11).



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  		Figure 4. DNA binding of in vitro translated HNF-4 to FXII promoter HNF-4-binding site. 5'-End-labeled FXII- and {alpha}1antitrypsin HNF-4 probes were incubated without (lanes 1 and 9) or with wheat-germ lysate-translated HNF-4 (lanes 3–8 and 11) in the amounts indicated, Mock-programmed lysate is included as a negative control (Ctrl; lanes 2 and 10). FXII promoter/HNF-4 complex is competitively inhibited by preincubating the reaction in the presence of 50-, 100-, and 250-fold excesses of unlabeled FXII HNF-4 probe (lanes 6–8).

 
These results indicate that HNF-4 directly interacts with specific binding sites present on FXII gene promoter, which may be required to elicit the antagonistic effect on ER{alpha}-dependent trans-activation.

Mutagenesis of HNF-4-binding sites on FXII gene promoter in NIH3T3
To evaluate the requirement of HNF-4 consensus sequences for repression of ER{alpha}-dependent induction of FXII promoter in liver vs. nonliver cells, the construct PT-CAT181 mut, containing mutations of both HNF-4-binding sites, was generated (see Materials and Methods). In transient transfection assays, in NIH3T3 cells, PT-CAT181 mut exhibited full estrogen responsiveness, with a magnitude comparable to that achieved by the native promoter (Fig. 5Go, A and B). The ER{alpha}-dependent induction of PT-CAT181 mut was not affected by modification of basal promoter activity, which remained very low in all experimental conditions tested. In contrast, abrogation of HNF-4-mediated repression was observed (Fig. 5BGo), indicating that integrity of HNF-4-binding sites is required to antagonize ER{alpha} trans-activation properties.

Similar results were obtained with the deletion construct PT-CAT{Delta}181, lacking nucleotides from +27 to +49, encompassing the downstream HNF-4 motif. Deletion of this HNF-4-binding site preserved estrogen inducibility, although at a lower magnitude compared with that of wild-type PT-CAT181 (10- vs. 30-fold, respectively). The lower estrogen induction exhibited by PT-CAT{Delta}181 was partially accounted for by an increased CAT activity in the presence of unliganded ER{alpha}. Site IV may therefore play an important role in eliciting the HNF-4 antagonistic effect.

These results indicate that HNF-4-repressive role in ER{alpha}-dependent trans-activation of FXII promoter strictly requires the interaction of the orphan receptor with its cognate promoter element.

Mutagenesis of HNF-4-binding sites on FXII gene promoter in HepG2 cells
The lower magnitude of ER{alpha} induction of FXII promoter in a liver-derived compared with a nonliver-derived cell environment and the lack of effect of exogenously transfected HNF-4 in modulating estrogen-dependent FXII promoter activity allowed us to hypothesize that in liver cells, endogenous HNF-4 exerts maximal antagonistic action.

In the attempt to reconstitute full estrogen responsiveness, transient transfection assays using the PT-CAT181 mut construct were performed in HepG2 cells. Figure 6Go shows a comparative analysis of the estrogen inducibility between the native PT-CAT181 and the PT-CAT181 mut constructs. As expected, the mutant FXII promoter, lacking both HNF-4-binding elements, exhibited a higher E2/ER-dependent induction compared with the wild-type promoter (~7- vs. ~4-fold induction, respectively). Similar reconstitution of estrogen responsiveness was obtained upon cotransfection of an expression vector for a dominant negative form of the HNF-4 protein, which lacks the entire DNA-binding domain (HNF-4DBD; Fig. 7Go). On the contrary, transfection of the expression vectors for HNF-4 or HNF-4DBD alone did not result in any significant modification of FXII basal promoter activity (Fig. 7Go).



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  		Figure 6. Comparative analysis of estrogen responsiveness of PT-CAT181 mut vs. native PT-CAT181 promoters in HepG2 cells. Native (PT-CAT181; left panel) and mutated (PT-CAT181 mut; right panel) FXII reporters (5 µg) were cotransfected with or without ER{alpha} (ER) in the absence (-E2) or presence (+E2) of E2. Transfection conditions and calculation of CAT activity are the same as described in Fig. 5Go. Results represent the average of four independent experiments, each performed in duplicate.

 
These results support the hypothesis of an active role played by endogenous HNF-4 in the context of a liver-derived cell environment in counteracting the trans-activation properties of ER{alpha}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HNF-4 has been traditionally considered an orphan transcription factor and a member of the superfamily of ligand-dependent nuclear receptors. Recently, long chain fatty acyl-coenzyme A thioesters have been identified as specific ligands of HNF-4, able to modulate its transcriptional activity depending upon their chain length and degree of saturation (32).

HNF-4 usually acts, alone or in combinatorial association with other tissue-specific or basal transcription factors, in promoting the transcription of a wide variety of target genes as well as of several coagulation factors (25, 33, 34, 35, 36, 37, 38, 39). In the present study we demonstrate that HNF-4 is instead capable of antagonizing ER{alpha} trans-activation properties on FXII gene promoter. To our knowledge this is the first study to demonstrate a repressive role of HNF-4 on ligand-activated transcription of a blood coagulation factor gene.

A transcription inhibitory role for HNF-4 has been recently demonstrated on the arginase gene, the promoter activity of which, either basal or stimulated by members of the CAAT/enhancer-binding protein family, is repressed by HNF-4, apparently through a DNA binding-independent mechanism (40). In addition, Murao et al. have recently shown that HNF-4 is able to bind to and, in the kidney-derived cell line BHK, to repress transcriptional activity of the cis-acting site A of rat apolipoprotein A1 promoter (41).

Our results imply the existence of a negative functional interaction between two members of the nuclear hormone receptor superfamily, the liver-enriched HNF-4 and ER{alpha}, which may be relevant in modulating estrogen-dependent transcription of the blood coagulation FXII gene. The specificity of the HNF-4 effect in inhibiting the liganded ER{alpha}-mediated induction of FXII promoter, but not that of vitellogenin promoter, rules out the possibility that the HNF-4 effect may be due to a general inhibition of transcription. Instead, the existence of distinct cis-acting elements mediating HNF-4 repression, which are present in the former, but not in the latter, estrogen-dependent gene, denotes the specificity of HNF-4-ER{alpha} functional interaction on FXII promoter activity. Indeed, in the context of FXII promoter, we identified two putative HNF-4-binding regions (sites III and IV) that are protected from DNase I digestion by the addition of nuclear extracts from liver-derived HepG2 cells, endogenously expressing high HNF-4 protein levels (27, 30, 31), but not by nuclear extracts of the nonliver NIH3T3 cell line. Direct binding of recombinant HNF-4 protein to the footprinted regions confirmed the specificity of HNF-4 interaction with FXII promoter (Fig. 4Go). Mutations of both sites III and IV heavily impaired the HNF-4 inhibitory potency, thus indicating that HNF-4 exerts its inhibitory function through a DNA-dependent mechanism.

In a liver-type environment, disruption of HNF-4-binding sites results in a higher estrogen-dependent inducibility of FXII promoter due to the abrogation of repression exerted by the endogenous HNF-4 protein. Similar results were obtained after the addition of an HNF-4 protein (HNF-4DBD) deleted in the DNA-binding domain and exhibiting dominant negative activity, which is able to counteract endogenous HNF-4 (22). The dominant negative activity of the HNF-4DBD mutant is different from that of other dominant negative nuclear receptors, which usually contain mutation in the ligand-binding domain (42). Deletion of the DBD eliminates DNA binding without altering the ability to dimerize with the wild-type protein (22). As homodimerization seems essential for HNF-4 function, the DBD mutant exhibits its dominant negative action by allowing the formation of defective dimers with the wild-type protein. These data, therefore, address a direct role for HNF-4 in modulating estrogen-dependent transcription of the FXII gene. Nevertheless, the magnitude of estrogen inducibility could not reach the levels obtained in NIH3T3 cells. This effect could be accounted for by the higher basal activity of the native FXII promoter observed in HepG2 cells (Ref. 8 and this paper) and/or the effect of other liver-specific factors that may contribute to regulate FXII gene promoter activity.

Several hypotheses may be raised concerning the mechanism underlying the negative functional interaction between HNF-4 and ER{alpha} in modulating FXII gene promoter activity.

Our findings are against the possibility that HNF-4 may compete with ER{alpha} for binding to ERE. The assumption is based on sequence similarities between the ERE and the HNF-4-binding site, recently classified as a direct repeat of the AGGTCA motif that preferentially binds HNF-4 homodimers (20). Indeed, it has been proposed that HNF-4 could bind to an overlapping sequence with the retinoic acid-responsive element in the phosphoenolpyruvate carboxykinase promoter (38), whereas the HNF-4 DNA-binding domain has been demonstrated to heterodimerize with other members of the nuclear receptor superfamily (43). However, the various HNF-4-binding sites identified to date show a relative flexibility in their nucleotide composition, as different motifs have been demonstrated to physically and functionally interact with HNF-4 protein. For example, the ACTTTG nucleotide sequence seems very important in binding HNF-4 and mediating its transcriptional stimulation of blood coagulation factor VII and X genes (44, 45). Despite structure similarities in their specific DNA-binding sites, a competition between HNF-4 and ER{alpha} in binding FXII ERE may be ruled out by the observation that in NIH3T3 cells, mutation of HNF-4-binding sites abolishes HNF-4 repression while not modifying the estrogen-dependent inducibility of FXII promoter. The absence of a direct competition between HNF-4 and ER{alpha} on ERE is also supported by the lack of HNF-4 in both NIH3T3 and HepG2 cells to impair the activity of vitellogenin promoter, which also contains a canonical ERE and is induced by estrogens.

Another mechanism by which HNF-4 may repress ER{alpha}-mediated induction of the FXII gene is through interference with the overall function of the basal transcription machinery. Indeed, TFIIB, a key component of the basal transcription complex, has been demonstrated to physically interact with HNF-4 in facilitating the assembly of the preinitiation complex on the apolipoprotein A1 gene promoter (27). In our model, a steric hindrance of HNF-4 to the ER{alpha}-stimulated transcriptional deployment of the RNA polymerase II complex could be hypothesized due to the unusual location of HNF-4-binding sites downstream from the FXII major transcription start site. However, the absence of HNF-4 effects in altering FXII basal promoter activity in either NIH3T3 or HepG2 cells challenges the possibility of interference with the function of the basal transcription machinery.

A third and more conceivable mechanism is based on the sequestration of a common accessory factor, which is present in HepG2 and NIH3T3 cells and is able to interact with both HNF-4 and ER{alpha}. Two potential candidates, the general transcription factor TFIIB and the cointegrator cAMP response element binding proteins, however, were excluded because their addition did not allow rescue of HNF-4 repression of estrogen-induced FXII gene expression (data not shown). Whether the putative accessory factor is represented by one of the recently identified nuclear hormone receptor coactivators (i.e. RIP-140, SRC1, etc.) or by a new, yet to be identified, factor remains to be verified. The lower estrogen inducibility as well as the lack of exogenous HNF-4 to affect ER{alpha}-induced FXII promoter activity in HepG2 cells may therefore be explained by the coexistence in this cell line of all necessary transcription regulatory elements. On the contrary, in NIH3T3 cells, which do not contain endogenous HNF-4, an unopposed and greater estrogen inducibility of FXII gene expression is observed. Hence, exogenous addition of the lacking HNF-4 component resulted in strong inhibition of estrogen-induced FXII promoter activity. For similar reasons the vitellogenin promoter, which instead lacks the required and essential HNF-4-binding sites, is unaffected by exogenous addition of HNF-4 in any of the cell lines tested.

The combinatorial interaction between liver-specific and ubiquitous, hormone-dependent transcription factors may therefore contribute to impart the tissue specificity of FXII gene expression, whereas the repressor function of HNF-4 on estrogen inducibility of the FXII gene may be involved in precise and fine regulation of expression of the blood coagulation factor. The recent discovery of long chain fatty acyl-coenzyme A thioesters as specific ligands of HNF-4 and their ability to modulate the affinity of the nuclear receptor for its cognate promoter elements, resulting in activation or inhibition of HNF-4 transcriptional activity (32) raise important implications for the pathogenesis of blood coagulability and fibrinolytic disorders as well as of other common diseases, such as cancer, atherogenesis, and diabetes.


   Acknowledgments
 
We thank Dr. Gavin Kelsey for the recombinant HNF-4-DBD, Dr. Todd Leff for the human HNF-4{alpha}1, Dr. Frances Sladek for recombinant HNF-4-expressing constructs and anti-HNF-4 antiserum, and Dr. Carlo Gaetano for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by research grants from Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Consiglio Nazionale delle Ricerche, Associazione Italiana per la Ricerca sul Cancro, Casa Sollievo Sofferenca Hospital, San Giovanni Rotondo, Italy. Back

Received February 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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