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Inhibitory Effect of the Small Heterodimer Partner on Hepatocyte Nuclear Factor-4 Mediates Bile Acid-induced Repression of the Human Angiotensinogen Gene

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m310577200

ISSN

1083-351X

Autores

Yoko Shimamoto, Junji Ishida, Kazuyuki Yamagata, Tomoko Saito, Hideki Kato, Toshiki Matsuoka, Keiko Hirota, Hiroaki Daitoku, Masaomi Nangaku, Kazuya Yamagata, Hiroshi Fujii, Jun Takeda, Akiyoshi Fukamizu,

Tópico(s)

Nuclear Receptors and Signaling

Resumo

Bile acids function as transcriptional regulators for the genes important in bile acid synthesis and cholesterol homeostasis. In this study, we identified angiotensinogen (ANG), the precursor of vasoactive octapeptide angiotensin II, as a novel target gene of bile acids. In human ANG transgenic mice, administration of cholic acid resulted in the down-regulation of human ANG gene expression in the liver. ANG gene expression in HepG2 cells was also repressed by chenodeoxycholic acid. Because the expression of small heterodimer partner (SHP) mRNA was induced by chenodeoxycholic acid in HepG2 cells, we analyzed the effects of SHP on the human ANG promoter. Promoter mutation analysis demonstrated that SHP repressed human ANG promoter activity through the element, which has been previously determined as a binding site for hepatocyte nuclear factor-4 (HNF-4). SHP repressed human ANG promoter activity only when the HNF-4 expression vector was cotransfected in HeLa cells. Furthermore, we found that SHP bound to the HNF-4 N-terminal region including the DNA-binding domain and activation function-1 and that SHP prevented HNF-4 from binding to the human ANG promoter. These results suggest that bile acids negatively regulate the human ANG gene through the inhibitory effect of SHP on HNF-4. Bile acids function as transcriptional regulators for the genes important in bile acid synthesis and cholesterol homeostasis. In this study, we identified angiotensinogen (ANG), the precursor of vasoactive octapeptide angiotensin II, as a novel target gene of bile acids. In human ANG transgenic mice, administration of cholic acid resulted in the down-regulation of human ANG gene expression in the liver. ANG gene expression in HepG2 cells was also repressed by chenodeoxycholic acid. Because the expression of small heterodimer partner (SHP) mRNA was induced by chenodeoxycholic acid in HepG2 cells, we analyzed the effects of SHP on the human ANG promoter. Promoter mutation analysis demonstrated that SHP repressed human ANG promoter activity through the element, which has been previously determined as a binding site for hepatocyte nuclear factor-4 (HNF-4). SHP repressed human ANG promoter activity only when the HNF-4 expression vector was cotransfected in HeLa cells. Furthermore, we found that SHP bound to the HNF-4 N-terminal region including the DNA-binding domain and activation function-1 and that SHP prevented HNF-4 from binding to the human ANG promoter. These results suggest that bile acids negatively regulate the human ANG gene through the inhibitory effect of SHP on HNF-4. Bile acids are physiological detergents that facilitate excretion, absorption, and transport of fats and sterols in the intestine and liver. It has been revealed recently that bile acids also function as signaling molecules for regulating bile acid metabolism and cholesterol homeostasis (1Chiang J.Y. Am. J. Physiol. 2003; 284: G349-356Crossref PubMed Scopus (131) Google Scholar). A major advance toward understanding the molecular mechanism of bile acid-induced negative feedback regulation of the genes involved in bile acid synthesis has come with the identification of the small heterodimer partner (SHP) 1The abbreviations used are: SHP, small heterodimer partner; HNF-4, hepatocyte nuclear factor-4; ANG, angiotensinogen; CDCA, chenodeoxycholic acid; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. as a negative nuclear receptor induced by the farnesoid X receptor, a nuclear receptor of bile acids (2Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. 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Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar), and the liver receptor homolog that activates the transcription of cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis (2Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 3Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar). Angiotensinogen (ANG), the precursor of vasoactive octapeptide angiotensin II, is mainly synthesized in the liver and secreted into the circulation (11Corvol P. Jeunemaitre X. Endocr. Rev. 1997; 18: 662-677PubMed Google Scholar). We have identified various regulatory elements and factors that regulate human ANG gene transcription (12Yanai K. 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Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 19Shimada S. Yanai K. Takahashi S. Murakami K. Fukamizu A. Endocrine. 1995; 3: 543-547Crossref PubMed Scopus (7) Google Scholar). Our previous studies have shown that cis-acting elements located at nucleotides –1222 to +44 are sufficient for human ANG gene expression in transiently transfected human hepatoma HepG2 cells and in the livers of transgenic mice (20Fukamizu A. Takahashi S. Seo M.S. Tada M. Tanimoto K. Uehara S. Murakami K. J. Biol. Chem. 1990; 265: 7576-7582Abstract Full Text PDF PubMed Google Scholar, 21Takahashi S. Fukamizu A. Hasegawa T. Yokoyama M. Nomura T. Katsuki M. Murakami K. Biochem. Biophys. Res. Commun. 1991; 180: 1103-1109Crossref PubMed Scopus (57) Google Scholar) and that HNF-4 plays an important role in hepatic ANG expression (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The human ANG transgenic mouse exhibits high blood pressure through cross-mating with the line expressing human renin (22Fukamizu A. Sugimura K. Takimoto E. Sugiyama F. Seo M.S. Takahashi S. Hatae T. Kajiwara N. Yagami K. Murakami K. J. Biol. Chem. 1993; 268: 11617-11621Abstract Full Text PDF PubMed Google Scholar). It has been shown that bile duct ligation in animals, an experimental model of liver cirrhosis, results in a decrease in blood pressure and a transient decrease in plasma ANG despite an increase in plasma renin activity (23Shasha S.M. Better O.S. Chaimovitz C. Doman J. Kishon Y. Clin. Sci. Mol. Med. 1976; 50: 533-537PubMed Google Scholar, 24Naveh Y. Finberg J.P. Kahana L. Better O.S. J. Hepatol. 1988; 6: 57-62Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 25Ubeda M. Matzilevich M.M. Atucha N.M. Garcia-Estan J. Quesada T. Tang S.S. Ingelfinger J.R. Hepatology. 1994; 19: 1431-1436Crossref PubMed Scopus (19) Google Scholar). Interestingly, hepatorenal syndrome, a disease with acute renal failure induced by severe hepatic diseases, is thought to result from the disruption of vasoactive factor control, but its mechanism is ill defined. In hepatorenal syndrome, arterial vasodilation occurs despite high plasma renin activity (26Gines P. Arroyo V. J. Am. Soc. Nephrol. 1999; 10: 1833-1839PubMed Google Scholar). These observations prompted us to investigate the effect of bile acids on ANG transcription. In this study, we measured ANG mRNA levels in human ANG transgenic mice and HepG2 cells, both treated with bile acids, and demonstrated that bile acids repress the expression of the human ANG gene. Furthermore, we demonstrate that SHP represses the human ANG promoter by inhibiting HNF-4 binding to the promoter. These results suggest that bile acids repress human ANG transcription through the effect of the negative nuclear receptor SHP on HNF-4. Animal Studies—Female human ANG transgenic mice (21Takahashi S. Fukamizu A. Hasegawa T. Yokoyama M. Nomura T. Katsuki M. Murakami K. Biochem. Biophys. Res. Commun. 1991; 180: 1103-1109Crossref PubMed Scopus (57) Google Scholar) in a C57BL/6 genetic background (2 months old) were treated for 7 days with experimental diets consisting of the control diet supplemented with 1% (w/w) cholic acid. At the end of the treatment period, all animals were fasted overnight, weighed, and killed by cervical dislocation. Livers were frozen in liquid nitrogen and stored at –80 °C until used. Cell Culture and Transfection Assays—HepG2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. HepG2 cells used in Northern blot analysis were plated at a density of 3 × 106 cells/100-mm dish; washed; incubated for 24 h in serum-free medium containing 5 μg/ml insulin, 5 μg/ml transferrin, 0.01% fatty acid-free bovine serum albumin, and 100 μm chenodeoxycholic acid (CDCA); and harvested for assays. HepG2 cells used in transfection assays were plated at a density of 5 × 104 cells/24-well cluster tissue culture plate. Transfections were performed using FuGENE 6 (Roche Applied Science). All samples were complemented with empty pcDNA3 vectors (Invitrogen) to an equal total amount of DNA (300 ng). After 48 h of incubation, luciferase activity was assayed with ARVO™SX (Wallac Berthold). Transfection efficiency was monitored by cotransfecting 100 ng of SV40-driven β-galactosidase expression plasmid. RNA Analysis—Total RNA was isolated from livers or HepG2 cells using ISOGEN (Nippon Gene) based on the acid guanidium thiocyanate/phenol/chloroform extraction method (27Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). For Northern blot analysis, 2 or 10 μg of RNA was denatured with glyoxal, separated by electrophoresis, and transferred onto a nylon membrane. The membrane was hybridized to 32P-labeled DNA probes at 60 °C for 15 h and washed twice with 2× SSC at room temperature for 5 min, twice with 2× SSC and 1% SDS at 65 °C for 15 min, and twice with 0.1× SSC at room temperature for 5 min. The DNA probes used were the 298-bp ApaI/EcoRI fragment from pHag3 (21Takahashi S. Fukamizu A. Hasegawa T. Yokoyama M. Nomura T. Katsuki M. Murakami K. Biochem. Biophys. Res. Commun. 1991; 180: 1103-1109Crossref PubMed Scopus (57) Google Scholar) for analysis of human ANG transgenic mice, the 413-bp RsaI fragment from phAG27B (20Fukamizu A. Takahashi S. Seo M.S. Tada M. Tanimoto K. Uehara S. Murakami K. J. Biol. Chem. 1990; 265: 7576-7582Abstract Full Text PDF PubMed Google Scholar) for analysis of HepG2 cells, the 722-bp EcoRI/SacI fragment from pcDNA3HA/hHNF-4α2 (28Hirota K. Daitoku H. Matsuzaki H. Araya N. Yamagata K. Asada S. Sugaya T. Fukamizu A. J. Biol. Chem. 2003; 278: 13056-13060Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), human SHP cDNA (29Nishizawa H. Yamagata K. Shimomura I. Takahashi M. Kuriyama H. Kishida K. Hotta K. Nagaretani H. Maeda N. Matsuda M. Kihara S. Nakamura T. Nishigori H. Tomura H. Moore D.D. Takeda J. Funahashi T. Matsuzawa Y. J. Biol. Chem. 2002; 277: 1586-1592Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and human β-actin cDNA (Clontech). Recombinant Plasmids—For the human ANG promoter-luciferase chimeric constructs, BglII/HindIII fragments from 13cat, DM4cat, DM7cat, DM7.8cat, DM8cat, and DM9cat (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) were subcloned into the BglII/HindIII sites of pGV-B2 (Wako). The 13ΔC-Luc construct was generated by PCR-based deletion (30Kuipers O.P. Boot H.J. de Vos W.M. Nucleic Acids Res. 1991; 19: 4558Crossref PubMed Scopus (89) Google Scholar) of nucleotides –429 to –386 using 13-Luc as a template, and the deleted fragment was subcloned into pGV-B2. For GST-SHP expression in Escherichia coli strains, the SHP cDNA fragments were generated by PCR and subcloned into pGEX-5X-1 (Amersham Biosciences). The HNF-4 deletion fragments were generated by PCR and subcloned into pcDNA3 tagged with the hemagglutinin epitope. pcDNA3/hSHP-HA (29Nishizawa H. Yamagata K. Shimomura I. Takahashi M. Kuriyama H. Kishida K. Hotta K. Nagaretani H. Maeda N. Matsuda M. Kihara S. Nakamura T. Nishigori H. Tomura H. Moore D.D. Takeda J. Funahashi T. Matsuzawa Y. J. Biol. Chem. 2002; 277: 1586-1592Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), pcDNA3HA/hHNF-4α2 (28Hirota K. Daitoku H. Matsuzaki H. Araya N. Yamagata K. Asada S. Sugaya T. Fukamizu A. J. Biol. Chem. 2003; 278: 13056-13060Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and CSVP-Luc (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) were constructed as described previously. GST Pull-down Assays—The GST fusion proteins of each SHP mutant were expressed in E. coli BL21 (Promega) and purified using glutathione-Sepharose beads (Amersham Biosciences) following the manufacturer's directions. [35S]Methionine-labeled HNF-4 and its mutants were prepared by in vitro translation using the TnT coupled reticulocyte lysate system (Promega) and T7 RNA polymerase. [35S]Methionine-labeled proteins were incubated with the GST fusion protein bound to glutathione-Sepharose beads at 4 °C for 4 h in 500 μl of binding buffer (20 mm HEPES (pH 7.9), 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Nonidet P-40, 10% glycerol, and protease inhibitors). After washing the beads with binding buffer, the pull-down complexes were fractionated by SDS-PAGE and analyzed using an imaging analyzer. HEK293T whole cell extracts transiently expressing FLAG-tagged HNF-4 were prepared with lysis buffer (0.1% Nonidet P-40, 150 mm KCl, 20 mm HEPES (pH 7.9), 1 mm dithiothreitol, and protease inhibitors). HEK293T whole cell extracts were incubated with GST fusion protein bound to glutathione-Sepharose beads at 4 °C for 5 h in 1 ml of lysis buffer. After washing the beads with lysis buffer, the pull-down complexes were fractionated by SDS-PAGE and electrotransferred onto polyvinylidene fluoride membrane (Millipore Corp.). After blocking with 5% nonfat milk in 20 mm Tris-HCl (pH 7.5), 137 mm NaCl, and 0.1% Tween 20, the membrane was probed with anti-FLAG antibody M2 and detected with enhanced chemiluminescence reagents (PerkinElmer Life Sciences). Electrophoretic Mobility Shift Assays—HepG2 nuclear extracts were prepared as described previously (31Yoshida E. Aratani S. Itou H. Miyagishi M. Takiguchi M. Osumu T. Murakami K. Fukamizu A. Biochem. Biophys. Res. Commun. 1997; 241: 664-669Crossref PubMed Scopus (94) Google Scholar). GST-SHP and GST were expressed in E. coli BL21; purified using glutathione-Sepharose beads; eluted with 50 mm Tris-HCl (pH 8.0) and 20 mm reduced glutathione; dialyzed against reaction buffer (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar); and concentrated using Aquacide II (Calbiochem). The C region double-stranded oligonucleotides were prepared as described previously (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). 1 μg of HepG2 nuclear extract was incubated with 100 ng of poly[d(I-C)] (Roche Applied Science), 20 μg of bovine serum albumin, and GST-SHP or GST at 4 °C for 30 min in the presence or absence of 1 μl of anti-HNF-4 antibody (Santa Cruz Biotechnology). Subsequently, the nuclear extract was incubated with 0.1 ng of end-labeled oligonucleotide at 4 °C for 30 min. The binding reaction was carried out as described previously (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The reaction mixtures were directly loaded onto 4.5% nondenaturing polyacrylamide gels containing 5% glycerol made in 1× 90 mm Tris-HCl (pH 8.0), 89 mm boric acid, and 2 mm EDTA. Electrophoresis was performed at 130 V for 3 h at 4 °C, and the gels were dried and analyzed with a bioimaging analyzer. Cholic Acid Decreases Liver ANG mRNA Levels in Human ANG Transgenic Mice—To assess whether bile acids affect the expression of the human ANG gene, human ANG transgenic mice were treated for 7 days with a 1% (w/w) cholic acid diet. Northern blot analysis was performed to measure human ANG mRNA levels in the liver, the major site of ANG production. A human ANG-specific probe (21Takahashi S. Fukamizu A. Hasegawa T. Yokoyama M. Nomura T. Katsuki M. Murakami K. Biochem. Biophys. Res. Commun. 1991; 180: 1103-1109Crossref PubMed Scopus (57) Google Scholar) was used to detect ANG mRNA, and β-actin mRNA levels were measured to normalize ANG mRNA expression. The administration of cholic acid resulted in an ∼50% decrease in human ANG gene expression compared with that in control chow diet-fed mice (Fig. 1A). We also measured the expression of HNF-4, which is a major regulator of human ANG gene transcription in the liver (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). HNF-4 mRNA levels did not change significantly in this experiment (Fig. 1B). CDCA Decreases Human ANG mRNA in HepG2 Cells—We further examined the effect of bile acids on ANG expression in human hepatoma HepG2 cells. Because the function of HepG2 cells transporting bile acids across cell membranes is markedly small compared with that of normal liver cells (32Kullak-Ublick G.A. Beuers U. Paumgartner G. Hepatology. 1996; 23: 1053-1060Crossref PubMed Google Scholar), we used CDCA, a hydrophobic bile acid that does not require transporters. HepG2 cells were incubated for 0, 12, and 24 h in medium containing 100 μm CDCA, and ANG mRNA levels were determined by Northern blot analysis. In the 24-h treated cells, there was an ∼70% reduction in the level of human ANG mRNA compared with that in vehicle-treated cells (Fig. 2A). Because SHP is induced by bile acids to repress target genes (2Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 3Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar), we measured the mRNA levels of SHP. SHP mRNA levels showed a 3.5-fold increase in the 12-h treated cells, and the levels were reduced to the same levels as in vehicle-treated cells 24 h after stimulation (Fig. 2B). These data indicate that bile acid induces SHP mRNA and represses ANG mRNA in HepG2 cells. SHP Represses Human ANG Promoter Activity in HepG2 Cells—We wondered whether the repression of the ANG gene by bile acids is caused by SHP induction. The effect of SHP on the 5′-deletions of the human ANG promoter was analyzed in HepG2 cells. As shown in Fig. 3A, SHP reduced the activity of the ANG promoter containing sequence –1222 to +44 (13-Luc). This repression was still observed in the DM4 and DM7 constructs, but was abolished in the DM7.8, DM8, and DM9 constructs. Therefore, sequence –516 to –344 appears to contain the site negatively regulated by SHP, which includes the HNF-4-responsive region (nucleotides –429 to –386) referred to as the C region (18Yanai K. Hirota K. Taniguchi-Yanai K. Shigematsu Y. Shimamoto Y. Saito T. Chowdhury S. Takiguchi M. Arakawa M. Nibu Y. Sugiyama F. Yagami K. Fukamizu A. J. Biol. Chem. 1999; 274: 34605-34612Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). To test whether the C region could mediate the repression by SHP, two types of reporters were used: the 13ΔC-Luc reporter (the C region was deleted from the –1222/+44 promoter construct) for Fig. 3B and the CSVP-Luc reporter (six copies of the C region were cloned in front of the SV40 promoter) for Fig. 3C. Whereas SHP repressed the 13-Luc reporter, it failed to repress the 13ΔC-Luc reporter (Fig. 3B). Furthermore, SHP repressed the CSVP-Luc reporter, whereas the control SV40 promoter (SVP-Luc) was not repressed by SHP cotransfection (Fig. 3C). These results indicate that the C region is the cis-acting element that is responsible for SHP repressing human ANG promoter activity. SHP Represses Human ANG Promoter Activity via HNF-4 — We tested whether SHP represses the human ANG gene promoter by inhibiting HNF-4 transcriptional activity. To this end, we assessed the effect of SHP on human ANG promoter activity in HeLa cells, which do not express endogenous HNF-4 (33Rajas F. Gautier A. Bady I. Montano S. Mithieux G. J. Biol. Chem. 2002; 277: 15736-15744Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Transfection of HNF-4 activated the transcription of luciferase driven by the ANG promoter in a dose-dependent manner. As expected, cotransfection of SHP with the HNF-4 expression vector resulted in a dose-dependent repression of ANG promoter activity. In contrast, SHP failed to repress ANG promoter activity without the cotransfection of the HNF-4 expression vector (Fig. 4A). Similarly, transfection of HNF-4 activated the transcription of the CSVP-Luc reporter, and cotransfection of SHP with the HNF-4 expression vector resulted in a dose-dependent repression of the CSVP-Luc reporter. SHP did not repress the activity of the CSVP-Luc reporter without the co-transfection of the HNF-4 expression vector. The control SV40-driven luciferase gene did not respond to the cotransfection of the HNF-4 or SHP expression vector (Fig. 4B). These results demonstrate that SHP prevents HNF-4 from activating the human ANG promoter. SHP Inhibits the Binding of HNF-4 to the Human ANG Promoter—To determine how SHP inhibits the transcriptional activity of HNF-4, we analyzed the interaction sites in HNF-4 and SHP. By GST pull-down assay, we confirmed that SHP binds to AF-2 (activation function-2) of HNF-4 as reported previously (10Lee Y.K. Dell H. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar), and demonstrated that it bound to the N-terminal region of HNF-4, which includes the DNA-binding domain and AF-1 (activation function-1) (Fig. 5A). Conversely, we confirmed that HNF-4 bound to the region of SHP located at amino acids 1–160 (SHP-NT), but not to the region at amino acids 161–257 (SHP-CT), by GST pull-down assay (Fig. 5B). Because SHP binds to the region of HNF-4 involved in DNA binding (34Hadzopoulou-Cladaras M. Kistanova E. Evagelopoulou C. Zeng S. Cladaras C. Ladias J.A. J. Biol. Chem. 1997; 272: 539-550Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 35Jiang G. Lee U. Sladek F.M. Mol. Cell. Biol. 1997; 17: 6546-6554Crossref PubMed Scopus (24) Google Scholar), we further assessed whether SHP affects the binding of HNF-4 to the C region. Electrophoretic mobility shift assay using the labeled human ANG C region oligonucleotide with HepG2 nuclear extract resulted in the formation of two retarded complexes (Fig. 5C, lane 1). The supershift experiment using anti-HNF-4 antibody showed that the major band represents the HNF-4 complex (lane 8). This binding was C region-specific because the unlabeled C region oligonucleotide efficiently competed for this binding (lane 9). The reaction of GST-SHP with the C region determined that SHP did not bind to the C region (lane 10). Addition of GST-SHP to HepG2 nuclear extract inhibited HNF-4 binding to the C region in a dose-dependent manner (lanes 5–7). Control GST protein did not affect the binding of HNF-4 to the C region (lanes 2–4). Furthermore, we confirmed that the SHP-NT mutant, which bound to HNF-4, inhibited the DNA binding of HNF-4 (Fig. 5D, lane 3). In contrast, the SHP-CT mutant, which did not bind to HNF-4, did not prevent HNF-4 binding to the C region (lane 4). These experiments demonstrate that SHP inhibits the binding of HNF-4 to the C region of the human ANG promoter. It is well known that bile acids are not only a simple metabolic by-product, but a transcriptional regulator of bile acid metabolism. The target genes for bile acids involved in bile acid metabolism, cholesterol homeostasis, and gluconeogenesis such as the cholesterol 7α-hydroxylase gene (CYP7A1) (2Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1531) Google Scholar, 3Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1232) Google Scholar), the phospholipid transfer protein gene (36Urizar N.L. Dowhan D.H. Moore D.D. J. Biol. Chem. 2000; 275: 39313-39317Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and the phosphoenolpyruvate carboxykinase gene (37De Fabiani E. Mitro N. Gilardi F. Caruso D. Galli G. Crestani M. J. Biol. Chem. 2003; 278: 39124-39132Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) have been identified. It also had been reported recently that kininogen, a peptide that functions as a vasodilation factor, is up-regulated by bile acids (38Zhao A. Lew J.L. Huang L. Yu J. Zhang T. Hrywna Y. Thompson J.R. de Pedro N. Blevins R.A. Pelaez F. Wright S.D. Cui J. J. Biol. Chem. 2003; 278: 28765-28770Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In this work, we examined the effect of bile acids on ANG expression in the liver. Bile acid treatments decreased human ANG mRNA levels both in vivo and in the cell line (Figs. 1 and 2) and repressed human ANG promoter activity through SHP by inhibiting the binding of HNF-4 to the ANG promoter (Figs. 3, 4, 5). These results demonstrate that ANG, a vasoactive factor, is a novel target of bile acids. Two types of mechanisms of bile acid-induced gene transcriptional repression have been reported. One mechanism via a phosphorylation signaling pathway, activated by bile acids, such as JNK/c-Jun provides a rapid response to target gene repression. The other mechanism via SHP gene induction by the bile acid farnesoid X receptor shows a slower response compared with the JNK/c-Jun pathway (1Chiang J.Y. Am. J. Physiol. 2003; 284: G349-356Crossref PubMed Scopus (131) Google Scholar). In our study, the repression of ANG by CDCA was a relatively slow process, requiring between 12 and 24 h (Fig. 2A). Because it has been reported that the peak of repression by the JNK/c-Jun pathway takes place 2 h after bile acid stimulation (39Gupta S. Stravitz R.T. Dent P. Hylemon P.B. J. Biol. Chem. 2001; 276: 15816-15822Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), it is unlikely that the repression of ANG over 24 h after bile acid stimulation occurs due to the JNK/c-Jun pathway. Therefore, it was suggested that ANG gene expression is repressed by bile acids in a farnesoid X receptor/SHP-mediated process. A recent study reported that SHP represses HNF-4 transcriptional activity through the two mechanisms: passive inhibition by competition with coactivators and active repression by SHP with an unknown cause (10Lee Y.K. Dell H. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar). These mechanisms are supported by the fact that SHP binds to the AF-2 region of HNF-4, which functions as a cofactor target (10Lee Y.K. Dell H. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar). In this work, we demonstrated that SHP bound not only to the AF-2 region, but also to the N-terminal region of HNF-4, and that SHP inhibited the binding of HNF-4 to DNA (Fig. 5). The DNA binding inhibition by SHP may result from SHP masking the DNA-binding domain of HNF-4. On the other hand, there is another possible mechanism to inhibit DNA binding. It had been reported that the AF-2 region also functions as a homodimerization domain of HNF-4 and that homodimer formation has a critical role in binding to DNA (34Hadzopoulou-Cladaras M. Kistanova E. Evagelopoulou C. Zeng S. Cladaras C. Ladias J.A. J. Biol. Chem. 1997; 272: 539-550Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 35Jiang G. Lee U. Sladek F.M. Mol. Cell. Biol. 1997; 17: 6546-6554Crossref PubMed Scopus (24) Google Scholar). Therefore, it is possible that SHP inhibits the DNA binding of HNF-4 by preventing HNF-4 homodimerization via interaction with the AF-2 region of HNF-4. Although it has already been reported that SHP inhibits the DNA binding of the retinoic acid receptor/retinoid X receptor (4Seol W. Choi H.S. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (446) Google Scholar) and the liver receptor homolog (40Kovacic A. Speed C.J. Simpson E.R. Clyne C.D. Mol. Endocrinol. 2003; 18: 252-259Crossref PubMed Scopus (30) Google Scholar), our study is the first to demonstrate that SHP inhibits the DNA binding of HNF-4. ANG is the substrate of renin, the first and rate-limiting enzyme of the renin/angiotensin system that plays an important role in the regulation of blood pressure. It had been shown that bile acids are isolated as a renin inhibitor (41Kokubu T. Hiwada K. Yamamura Y. Hayashi K. Okumura J. Hori M. Kobayashi S. Ueno H. Biochem. Pharmacol. 1972; 21: 209-217Crossref PubMed Scopus (14) Google Scholar). In addition to this enzymatic inhibitory activity, this work, which identified ANG as a target gene for bile acids, provides another possible action point where bile acids negatively regulate the renin/angiotensin system at the transcriptional level. We thank Drs. Makoto Makishima and Ichiro Takada for helpful suggestions. We thank members of the Fukamizu laboratory for sharing unpublished data and critical comments.

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