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• W2005456200 abstract "Microsomal triglyceride transfer protein (MTP) is involved in the transfer of triglycerides, cholesterol esters, and phospholipids to newly synthesized apolipoprotein (apo) B. It is therefore essential for lipoprotein synthesis and secretion in the liver and the small intestine. Although several recent experiments have revealed the transcriptional regulation of the MTP gene, little has been revealed to date about hepatocyte nuclear factor-4 (HNF-4)-dependent regulation. We here report that the human MTP gene promoter contains a pair of functional responsive elements for HNF-4 and HNF-1, the latter of which is another target gene of HNF-4. Chromatin immunoprecipitation assays provide evidence that endogenous HNF-4 and HNF-1 can bind these elements in chromatin. In Hep G2 cells overexpression of either a dominant negative form of HNF-4 or small interfering RNAs (siRNAs) against HNF-4 dramatically reduces the activities of both the wild type and the HNF-4 site mutant MTP promoter. This suggests that HNF-4 regulates MTP gene expression either directly or indirectly through elevated HNF-1 levels. When Hep G2 cells were cultured with chenodeoxycholic acid (CDCA), a ligand for the farnesoid X receptor (FXR), mRNA levels for MTP and apo B were reduced because of increased expression of the factor small heterodimer partner (SHP), which factor suppresses HNF-4 activities. Chenodeoxycholic acid, but not a synthetic FXR ligand, attenuated expression of HNF-4, bringing about a further suppression of MTP gene expression. Over time the intracellular MTP protein levels and apo B secretion in the culture medium significantly declined. These results indicate that two nuclear receptors, HNF-4 and FXR, are closely involved in MTP gene expression, and the results provide evidence for a novel interaction between bile acids and lipoprotein metabolism. Microsomal triglyceride transfer protein (MTP) is involved in the transfer of triglycerides, cholesterol esters, and phospholipids to newly synthesized apolipoprotein (apo) B. It is therefore essential for lipoprotein synthesis and secretion in the liver and the small intestine. Although several recent experiments have revealed the transcriptional regulation of the MTP gene, little has been revealed to date about hepatocyte nuclear factor-4 (HNF-4)-dependent regulation. We here report that the human MTP gene promoter contains a pair of functional responsive elements for HNF-4 and HNF-1, the latter of which is another target gene of HNF-4. Chromatin immunoprecipitation assays provide evidence that endogenous HNF-4 and HNF-1 can bind these elements in chromatin. In Hep G2 cells overexpression of either a dominant negative form of HNF-4 or small interfering RNAs (siRNAs) against HNF-4 dramatically reduces the activities of both the wild type and the HNF-4 site mutant MTP promoter. This suggests that HNF-4 regulates MTP gene expression either directly or indirectly through elevated HNF-1 levels. When Hep G2 cells were cultured with chenodeoxycholic acid (CDCA), a ligand for the farnesoid X receptor (FXR), mRNA levels for MTP and apo B were reduced because of increased expression of the factor small heterodimer partner (SHP), which factor suppresses HNF-4 activities. Chenodeoxycholic acid, but not a synthetic FXR ligand, attenuated expression of HNF-4, bringing about a further suppression of MTP gene expression. Over time the intracellular MTP protein levels and apo B secretion in the culture medium significantly declined. These results indicate that two nuclear receptors, HNF-4 and FXR, are closely involved in MTP gene expression, and the results provide evidence for a novel interaction between bile acids and lipoprotein metabolism. MTP, 1The abbreviations used are: MTP, microsomal triglyceride transfer protein; VLDL, very low density lipoprotein; apo, apolipoprotein; HNF, hepatocyte nuclear factor; SHP, small heterodimer partner; FXR, farnesoid X receptor; RXR, retinoid X receptor; MAPK, mitogen-activated protein kinase; CDCA, chenodeoxycholic acid; LPDS, lipoprotein-deficient serum; FBS, fetal bovine serum; DN, dominant negative; siRNA, small interfering RNA; I-BABP, intestinal bile acid-binding protein; ChIP, chromatin immunoprecipitation; ERK, extracellular signal-regulated kinase; DR1, direct repeat 1. expressed specifically in the liver and the small intestine, plays a critical role in the assembly and secretion of very low density lipoproteins (VLDLs) and chylomicrons. MTP exists in the lumen of the endoplasmic reticulum as a heterodimer with protein-disulfide isomerase and is involved in the transfer of triglycerides, cholesterol esters, and phospholipids to newly synthesized apo B (1Jamil H. Dickson J.K. Chu C.-H. Lago M.W. Rinehart J.K. Biller S.A. Gregg R.E. Wetterau J.R. J. Biol. Chem. 1995; 270: 6549-6554Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 2Kulinski A. Rustaeus S. Vance J.E. J. Biol. Chem. 2002; 277: 31516-31525Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). If the apo B protein is not properly folded or if the enrichment of lipids is insufficient, the apo B protein is degraded by a ubiquitin-dependent proteasome process instead of proceeding to the formation of lipoprotein particles (3Davis R.A. Thrift R.N. Wu C.C. Howell K.E. J. Biol. Chem. 1990; 265: 10005-10011Abstract Full Text PDF PubMed Google Scholar, 4Sato R. Imanaka T. Takatsuki A. Takano T. J. Biol. Chem. 1990; 265: 11880-11884Abstract Full Text PDF PubMed Google Scholar, 5Fisher E.A. Ginsberg H.N. J. Biol. Chem. 2002; 277: 17377-17380Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). In human patients with abetalipoproteinemia the absence of functional MTP results in a defect in the assembly and secretion of plasma lipoproteins containing apo B (6Wettreau J.R. Aggerbeck L.P. Bouma M.-E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (642) Google Scholar). A vital role of MTP in the formation and secretion of apo B is further supported by the fact that conditional gene knock-out mice specifically lacking hepatic MTP are unable to form VLDLs in the liver (7Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Investig. 1999; 103: 1287-1298Crossref PubMed Scopus (362) Google Scholar). Moreover, specific inhibitors of MTP lipid transfer have been developed and have lowered plasma cholesterol levels successfully (8Wetterau J.R. Gregg R.E. Harrity T.W. Arbeeny C. Cap M. Connolly F. Chu C.H. George R.J. Gordon D.A. Jamil H. Jolibois K.G. Kunselman L.K. Lan S.J. Maccagnan T.J. Ricci B. Yan M. Young D. Chen Y. Fryszman O.M. Logan J.V. Musial C.L. Poss M.A. Robl J.A. Simpkins L.M. Slusarchyk W.A. Sulsky R. Taunk P. Magnin D.R. Tino J.A. Lawrence R.M. Dickson J.K. Biller S.A. Science. 1998; 282: 751-754Crossref PubMed Scopus (251) Google Scholar). These findings clearly indicate that changes in MTP activities under various physiological conditions can modulate lipoprotein production and secretion in the liver and intestine. HNF-4 is a highly conserved member of the nuclear receptor superfamily. It is a liver-enriched transcription factor that, together with other factors, plays a key role in the tissue-specific expression of a large number of genes involved in lipid and glucose metabolism. The active form of HNF-4 is a homodimer, and it does not appear to heterodimerize with other members of the nuclear receptor family. Recent investigations have shown that coenzyme A derivatives of certain fatty acids activate the receptor, and these derivatives thus have been characterized as endogenous ligands for HNF-4 (9Hertz R. Mangeheim J. Berman I. Bar-Tana J. Nature. 1998; 392: 512-516Crossref PubMed Scopus (455) Google Scholar, 10Petrescu A.D. Hertz R. Bar-Tana J. Schroeder F. Kier A.B. J. Biol. Chem. 2002; 277: 23988-23999Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A crucial role for HNF-4 in metabolic homeostasis was demonstrated by the finding that mutations in the HNF-4 gene cause the disorder known as maturity onset diabetes of the young (11Yamagata K. Furuta H. Oda N. Kaisaki P.J. Menzel S. Cox N.J. Fajans S.S. Signorini S. Stoffel M. Bell G.I. Nature. 1996; 384: 458-460Crossref PubMed Scopus (1058) Google Scholar). Conditional HNF-4 gene knock-out mice, which were produced using the Cre-loxP method with an albumin-Cre transgene, exhibit a great reduction in serum cholesterol and triglycerides because of the decreased levels of MTP and several apolipoproteins (12Hayhurst G.P. Lee Y.-H. Lambert G. Ward J.M. Gonzalez F.J. Mol. Cell. Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (867) Google Scholar). Although this result indicates that MTP gene expression is under the control of HNF-4, little is known about the specific step. It has been shown that the transcriptional activity of HNF-4 is regulated by interaction with small heterodimer partner (SHP), an atypical negative nuclear receptor lacking a DNA-binding domain (13Seol W. Choi H.S. Moore D.D. Science. 1996; 272: 1336-1339Crossref PubMed Scopus (446) Google Scholar, 14Lee 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). SHP, induced by FXR together with bile acids, controls the transcriptional activity of several other nuclear receptors including the constitutive androstane receptor, thyroid receptor, retinoid X receptor (RXR), retinoic acid receptor, estrogen receptors, peroxisome proliferator-activated receptors, the liver X receptor, and the liver receptor homolog-1 (14Lee 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, 15Seol W. Hanstein B. Brown M. Moore D.D. Mol. Endocrinol. 1998; 12: 1551-1557Crossref PubMed Scopus (0) Google Scholar, 16Johansson L. Thomsen J.S. Damdimopoulou A.E. Spyrou G. Gustafsson J.A. Treuter E. J. Biol. Chem. 1999; 274: 345-353Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17Masuda N. Yasumo H. Tamura T. Hashiguchi N. Furusawa T. Tsukamoto T. Sadano H. Osumi T. Biochim. Biophys. Acta. 1997; 1350: 27-32Crossref PubMed Scopus (48) Google Scholar, 18Goodwin 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 (1528) Google Scholar, 19Brendel C. Schoonjans K. Botrugno O.A. Treuter E. Auwerx J. Mol. Endocrinol. 2002; 16: 2065-2076Crossref PubMed Scopus (167) Google Scholar). Moreover, recent findings have provided evidence that bile acids activate a MAPK pathway (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Wang L. Lee Y.-K. Bundman D. Han Y. Thevananther S. Kim C.-S. Chua S.S. Wei P. Heyman R.A. Karin M. Moore D.D. Dev. Cell. 2002; 2: 721-731Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) and reduce the transactivation potential of HNF-4 (22De Fabiani E. Mitro N. Anzulovich A.C. Pinelli A. Galli G. Crestani M. J. Biol. Chem. 2001; 276: 30708-30716Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). These findings prompted us to examine the effect of bile acids, which activate FXR and eventually induce SHP gene expression, on the HNF-4-dependent transcription of the MTP gene. We here show that MTP gene expression is regulated by HNF-4 and HNF-1, which bind to the individual responsive element in the promoter of the human MTP gene. We also demonstrate that bile acids can down-regulate MTP transcription by impairing the transactivation potential of HNF-4 through the interaction with SHP and suppressing HNF-4 gene expression. In response to attenuated HNF-4 activity the transcription of other HNF-4-responsive genes including HNF-1 and apo B is also reduced, leading to decreased VLDL secretion. Taken together, this evidence suggests that bile acids are able to control lipoprotein synthesis and secretion via the FXR- and HNF-4-mediated pathways. Materials—CDCA, anti-FLAG M2 antibody, and lipoprotein-deficient serum (LPDS) were purchased from Sigma. An FXR ligand, GW4064, was custom synthesized. Cell Culture—Hep G2 and HEK293 cells were cultured with a medium containing 10% fetal bovine serum (FBS) in collagen-coated dishes as described previously (23Sato R. Okamoto A. Inoue J. Miyamoto W. Sakai Y. Emoto N. Shimano H. Maeda M. J. Biol. Chem. 2000; 275: 12497-12502Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Misawa K. Horiba T. Arimura N. Hirano Y. Inoue J. Emoto N. Shimano H. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 36176-36182Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Construction of Plasmids—Expression constructs of human HNF-4α2, pHNF4, and pFLAG-HNF4, were described previously (24Misawa K. Horiba T. Arimura N. Hirano Y. Inoue J. Emoto N. Shimano H. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 36176-36182Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). An expression construct of a dominant negative (DN) form of HNF-4, pDN-HNF4, which lacks the 110 amino acids from Arg2 to Gln111, was made from pHNF4. To generate plasmids of human HNF-1α (pHNF1) and SHP (pSHP), 1.9- and 1.8-kb fragments obtained by reverse transcription-PCR using total RNA from Hep G2 cells were ligated into a pME18S vector (25Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), respectively. An expression plasmid pGal4-HNF4 was constructed by inserting the fragment encoding human HNF-4α2 into a Gal4 DNA-binding domain expression vector, pM (Clontech). Expression constructs for human FXR, RXRα, and siRNA (pSiHNF4 and pSi) were described previously (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Misawa K. Horiba T. Arimura N. Hirano Y. Inoue J. Emoto N. Shimano H. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 36176-36182Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Construction of the Reporter Genes for Luciferase Assays—The reporter plasmid containing human MTP promoter (–204 to +33), pMTP-204, was described previously (25Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). All of the mutant reporter constructs (pMTP-AKO, -BKO, and -CKO and pMTPΔHNF1) were synthesized by a PCR-assisted method using a site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. All of the mutant HNF-4 and HNF-1 sites were replaced by the sequence shown by the italicized letters in Fig. 1. To generate pHNF4-1000, an 1-kb fragment containing the 5′-flanking region of the human gene (–1014 to +55) was inserted into a pGL3 basic vector (Promega). The reporter plasmid containing human intestinal bile acid-binding protein (I-BABP) promoter (–862 to +30), pI-BABP, was described previously (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Reporter Assays—Reporter assays were performed as described previously (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 26Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 27Hirano Y. Murata S. Tanaka K. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 16809-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). HEK293 cells were transfected with 0.2 μg of the indicated reporter construct, 0.01 μg of pRL-CMV, an expression plasmid encoding Renilla luciferase (Promega), and 0.6 μg of either pHNF4 or pHNF1. Hep G2 cells were transfected with 2 μg of the indicated reporter construct and 0.1 μg of pRL-CMV. In reporter assays in Fig. 8, Hep G2 cells were cultured with a medium containing 10% charcoalstripped FBS (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). After incubation for 48 h, the Dual-Luciferase™ reporter system (Promega) was used to determine luciferase activity. When Hep G2 cells were transfected with siRNA expression constructs, cells were harvested after 72 h of incubation. Gel Mobility Shift Assay—Gel mobility shift assays were performed as described previously (24Misawa K. Horiba T. Arimura N. Hirano Y. Inoue J. Emoto N. Shimano H. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 36176-36182Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To obtain recombinant HNF-4, HNF-1, and DN-HNF-4, HEK293 cells were transfected with one of the expression constructs, pFLAG-HNF4, pHNF1, or pDN-HNF4, and then their nuclear extracts were prepared. 32P-Labeled DNA probes (5′-GCGCTGGGCAAAGGTCACCTGC-3′) containing a consensus HNF-4 binding sequence (28Ueda A. Takeshita F. Yamashiro S. Yoshimura T. J. Biol. Chem. 1996; 271: 19339-19347Google Scholar), (5′-CTGCAGCCCACCTACGTTTAATCATTAATAGTGAGCCCTTCAG-3′) corresponding to the HNF-1 binding site of the human MTP gene, (5′-AGTGACTGGTTACTCTTTAACGTATCCAC-3′) containing a consensus HNF-1 binding sequence, and (5′-GATTTTGGAGTTTGGAGTCTGACCTTTC-3′) corresponding to the HNF-4 B site in the human MTP promoter were used. In competition assays, excess amounts of unlabeled fragments (30-, 100-, or 300-fold) were added prior to addition of the labeled probe. Northern Blot Analysis—Hep G2 cells were set up on day 0 in medium A (Dulbecco's modified Eagle's medium, 100 units/ml penicillin, and 100 μg/ml streptomycin) supplemented with 10% charcoal-stripped fetal calf serum. On day 1, the medium was removed, and the cells were then washed with phosphate-buffered saline and refed with medium A containing 5% LPDS supplemented with the indicated concentration of CDCA. After a 6-, 12-, 18-, or 24-h culture, total RNA was extracted, and Northern blotting was performed as described previously (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Hirano Y. Murata S. Tanaka K. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 16809-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A 670-bp fragment from human MTP, a 640-bp fragment from human apo B, a 980-bp fragment from human HNF-4α, a 770-bp fragment from human SHP, a 1900-bp fragment of human HNF-1α, and a 700-bp fragment from 36B4 were used as templates for 32P-labeled probes. Western Blot Analysis—Hep G2 cells were cultured as described above. On day 1, the cells were refed with medium A containing 5% LPDS supplemented with 100 or 200 μm CDCA. On day 5, the cells were harvested, and Western blot analysis was carried out using a polyclonal antibody against human MTP (RS001, Ref. 25Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). To analyze apo B secretion, on day 4, the cells were refed with the same medium, and then the culture medium was collected on day 5. Western blot analysis was performed using a polyclonal antibody against human apo B (Chemicon International Inc.) as described previously (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Hirano Y. Murata S. Tanaka K. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 16809-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Chromatin Immunoprecipitation (ChIP) Assays—Hep G2 cells were fixed with 1% formaldehyde in phosphate-buffered saline at 37 °C for 10 min, lysed, and sonicated (29Arimura N. Horiba T. Imagawa M. Shimizu M. Sato R. J. Biol. Chem. 2004; 279: 10070-10076Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Soluble chromatin prepared with a ChIP assay kit (Upstate Biotechnology) was immunoprecipitated with antibodies against acetyl histone H3 (Upstate Biotechnology), human HNF-4α (Santa Cruz Biotechnology, sc-8987), human HNF-1 (Santa Cruz Biotechnology, sc-8986), or the Gal4 DNA-binding domain (Santa Cruz Biotechnology, sc-575). Purified samples were used as templates for PCR performed for 44 cycles. Oligonucleotide primers composed of the sequences 5′-GTGAGAGACTGAAAACTGCAGC-3′ and 5′-CATCCAGTGCCCAGCTAGGAG-3′ (205 bp) for MTP (upstream and downstream) were used for PCR. HNF-4-dependent Regulation of the Human MTP Promoter— As it has been reported that all of the putative positive and negative response elements for the liver-specific MTP gene expression are localized within the human MTP promoter –142 bp region (30Hagan D.L. Kienzle B. Jamil H. Hariharan N. J. Biol. Chem. 1994; 269: 28737-28744Abstract Full Text PDF PubMed Google Scholar), we focused on the promoter activity of the first 200 bp 5′ to the transcription start site to identify the cis acting element for HNF-4. We found three putative HNF-4-responsive elements in this region (Fig. 1) and performed luciferase assays using wild type or mutant versions of reporter genes to confirm certain functional element(s) among them. When Hep G2 cells, which endogenously express HNF-4, were transfected with one of the reporter genes, the luciferase activities were significantly decreased only by disruption of the B site (Fig. 2A). When HEK293 cells were transfected, the promoter activity was undetectable unless an HNF-4 expression plasmid was introduced, suggesting that HNF-4 is responsible for cell type-specific expression of the MTP gene (Fig. 2B). Only mutation at the B site elicited a tremendous suppression of luciferase activities in the presence of HNF-4. These results indicate that the B site is important for the HNF-4-dependent induction of the MTP promoter activity. HNF-4 Can Bind to the B Site in the Human MTP Promoter— Next, gel mobility shift assays were performed to demonstrate direct binding of HNF-4 to the B site. Fig. 3 shows that a nucleotide probe that carried the binding sequence for HNF-4 (28Ueda A. Takeshita F. Yamashiro S. Yoshimura T. J. Biol. Chem. 1996; 271: 19339-19347Google Scholar) was shifted after the addition of nuclear extracts of HEK293 cells transfected with an expression plasmid for FLAG-tagged HNF-4 and was supershifted by the addition of anti-FLAG antibodies (lanes 1–3). Excess amounts of unlabeled A site or C site probes (wild type and mutant forms) did not affect the formation of DNA-HNF-4 complex (Fig. 3, lanes 4–6 and lanes 10–12). However, the addition of excess amounts of an unlabeled B site probe (wild type), but not the mutant variant, inhibited the formation of the complex (Fig. 3, lanes 7–9), suggesting that the –49 to –61 region is a binding site for HNF-4. These results are in good accord with the results in Fig. 2. HNF-1-dependent Regulation of the Human MTP Promoter— Because there exists a putative HNF-1-responsive element in the 200-bp promoter region (Fig. 1), we examined whether this site is functional. Reporter assays showed that the luciferase activities in Hep G2 cells transfected with a mutant version of reporter gene lacking the HNF-1-responsive element were much lower than those with a wild type reporter gene (Fig. 4A). To determine whether HNF-1 can bind to the putative HNF-1-responsive element, gel shift assays were performed with a nucleotide probe covering this element. A specific protein-DNA complex, which was not detected with the nuclear extracts from mock-transfected cells (Fig. 4B, lane 1), was detected in the presence of the nuclear extracts from FLAG-HNF-1-expressing cells (lane 2). This band was migrated to the same position as a control complex with a probe containing a consensus HNF-1 binding sequence (Fig. 4B, lane 6). The addition of excess amounts of unlabeled wild type probe (Fig. 4B, lanes 3 and 4) inhibited the formation of the complex, whereas the addition of unlabeled mutant probe did not affect it (lane 5). These results clearly indicate that the –98 to –111 region is a binding site for HNF-1 and crucial for the MTP promoter activity. To investigate whether both endogenous HNF-1 and -4 bind to the MTP promoter, we performed chromatin immunoprecipitation. As shown in Fig. 4C, endogenous HNF proteins bound to the promoter of the MTP gene (second and third lanes). An unrelated anti-Gal4 DNA-binding domain antibody did not generate any PCR products (Fig. 4C, fourth lane). These data indicate that MTP gene expression is driven by the binding of endogenous HNF-1 and -4 to the individual responsive element in chromatin. Endogenous HNF-4 Plays a Crucial Role for MTP Gene Expression—Although Figs. 2 and 3 clearly show that the MTP promoter activity requires both the HNF-4 and HNF-1 functions, we do not know how these factors coordinately regulate MTP gene expression. HNF-1 has been shown to be a direct transcriptional target of HNF-4 in liver (31Kuo C. Conley P.B. Chen L. Sladek F.M. Darnell J.E. Crabtree G.R. Nature. 1992; 355: 457-461Crossref PubMed Scopus (370) Google Scholar). Therefore, one reasonable hypothesis is that HNF-4 stimulates MTP gene expression indirectly by increasing HNF-1 levels, which in turn activates the MTP promoter. To confirm the role of HNF-4, we constructed an expression plasmid for DN-HNF-4 that possesses a functional domain but lacks a DNA-binding domain and therefore suppresses the activity of endogenous HNF-4. As shown in gel mobility shift assays (Fig. 5A), a complex of HNF-4 and a probe containing the B site in the MTP promoter (lane 1) was replaced by the addition of excess amounts of DN-HNF-4 (lane 4), suggesting that this dominant negative form suppresses the activity of HNF-4. To distinguish a direct action of HNF-4 from an indirect action mediated through HNF-1, we compared the activity of wild type and mutant promoters in the presence or absence of DN-HNF-4 in Hep G2 cells (Fig. 5B). The activities of wild type and ΔHNF1 promoters significantly declined by expression of DN-HNF-4 through the intact B site. Furthermore, expression of DN-HNF-4 led to reduction in the activity of the promoter with the mutation in the HNF-4 site, confirming that HNF-4 also stimulates MTP gene expression through an indirect effect via HNF-1. Alternatively, endogenous HNF-4 functions were repressed by specific siRNAs, which had already been shown to be effective in our previous report (24Misawa K. Horiba T. Arimura N. Hirano Y. Inoue J. Emoto N. Shimano H. Shimizu M. Sato R. J. Biol. Chem. 2003; 278: 36176-36182Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). It turns out that quite similar results were obtained by both methods, i.e. weakening of endogenous HNF-4 activities (Fig. 5, B and C). Although in these assay systems it is difficult to compare quantitatively the direct with the indirect action of HNF-4, the significant effects of DN-HNF-4 and siRNAs clearly indicate that both actions are substantial. CDCA Affects MTP Gene Expression—It has been shown that CDCA induces SHP gene expression through activation of FXR and that SHP in turn inactivates HNF-4 functions (14Lee 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). Taking into account the fact that apo B is also one of the target genes for HNF-4, it might be that bile acids reduce MTP-mediated secretion of apo B-containing lipoproteins from hepatocytes. To investigate this possibility, Hep G2 cells were cultured with a medium containing CDCA for 24 h, and Northern blot analyses were carried out. Because of a long cascade from FXR to MTP through SHP and HNF-4, we analyzed changes in mRNA levels for several genes up to 24 h so as not to overlook any effects of CDCA (Fig. 6). In addition, because we had demonstrated previously that CDCA activates the MAPK pathway (20Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), the cells were cultured with the indicated CDCA concentration to stimulate this pathway. As shown in Fig. 6, after a 6-h incubation, only mRNA levels for SHP, a target gene of FXR, were upregulated, whereas the others were unaffected. MTP mRNA levels were reduced after 12 h and longer incubation with 100 μm CDCA, and the levels were reduced more robustly with 200 μm CDCA. Similar patterns were observed in terms of apo B, HNF-1, and HNF-4, which are all direct transcriptional targets of HNF-4. These results imply that the FXR-mediated activation of SHP might lead to suppression of HNF-4 target gene expression. It is likely that SHP mRNA levels were reduced after 24 h because of a self-regulatory mechanism (32Lu T.T. Makishima M. Repa J.J. Schoonjas K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). SHP Suppresses Transcription of the MTP Gene—To investigate the direct effect of SHP on MTP promoter activity, luciferase assays were performed. When Hep G2 cells were transfected with an SHP expression plasmid, the MTP promoter activity was reduced in a dose-dependent manner (Fig. 7A). It appears that overexpressed SHP directly inhibits HNF-4 activities. To confirm an SHP-mediated reduction of HNF-4 activit" @default.
• W2005456200 created "2016-06-24" @default.
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