Disruption of TR4 Orphan Nuclear Receptor Reduces the Expression of Liver Apolipoprotein E/C-I/C-II Gene Cluster
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m304088200
ISSN1083-351X
AutoresEungseok Kim, Shaozhen Xie, Shauh-Der Yeh, Yi-Fen Lee, Loretta L. Collins, Yueh‐Chiang Hu, Chih-Rong Shyr, Xiao-Min Mu, Ning-Chun Liu, Yen-Ta Chen, Peng‐Hui Wang, Chawnshang Chang,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoApolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism. Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism. Apolipoprotein E (apoE) 1The abbreviations used are: apoapolipoproteinHCRhepatic control regionTR4TR4 orphan nuclear receptorTR4RETR4 response elementTR4KOTR4 knockoutRXRretinoid X receptorFXRfarnesoid X receptorDRdirect repeatIRinverted repeatHNF-4hepatic nuclear factor 4LucluciferaseTKthymidine kinaseHREhormone response elementmtmutantRTreverse transcriptase. is primarily synthesized in the liver, although it is widely expressed in various tissues (1Driscoll D.M. Getz G.S. J. Lipid Res. 1984; 25: 1368-1379Abstract Full Text PDF PubMed Google Scholar, 2Elshourbagy N.A. Liao W.S. Mahley R.W. Taylor J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 203-207Crossref PubMed Scopus (395) Google Scholar). ApoE is an important constituent of plasma lipoproteins, such as very low density lipoprotein and chylomicrons, and serves as a ligand for the receptor-mediated uptake of these lipoproteins by the liver (3Mahley R.W. Science. 1988; 240: 622-663Crossref PubMed Scopus (3395) Google Scholar). ApoE is involved in the pathogenesis of atherosclerosis through the modulation of cholesterol efflux from macrophages (4Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar), and liver-derived apoE also has access to arterial intima and induces regression of atherosclerotic lesions (5Tsukamoto K. Tangirala R. Chun S.H. Pure E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar). apolipoprotein hepatic control region TR4 orphan nuclear receptor TR4 response element TR4 knockout retinoid X receptor farnesoid X receptor direct repeat inverted repeat hepatic nuclear factor 4 luciferase thymidine kinase hormone response element mutant reverse transcriptase. ApoE expression has been shown to be modulated by tissue-specific enhancers in different tissues. The nuclear receptor liver X receptor/retinoid X receptor α (RXRα) heterodimer has been reported to regulate expression of the apoE/C-I/C-IV/C-II gene cluster in macrophages by binding to direct repeat (DR) 4 sequences in multienhancer domains located within the gene cluster (6Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (576) Google Scholar, 7Mak P.A. Laffitte B.A. Desrumaux C. Joseph S.B. Curtiss L.K. Mangelsdorf D.J. Tontonoz P. Edwards P.A. J. Biol. Chem. 2002; 277: 31900-31908Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Previous studies using transgenic mice show that in liver, the major source of plasma apoE, the expression of apoE, apoC-I, apoC-IV, and apoC-II are promoted by liver-specific enhancers called hepatic control regions (HCR-1 and HCR-2) (8Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar, 9Allan C.M. Walker D. Taylor J.M. J. Biol. Chem. 1995; 270: 26278-26281Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Allan C.M. Taylor S. Taylor J.M. J. Biol. Chem. 1997; 272: 29113-29119Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). However, the molecular mechanism controlling HCR regulation of apoE expression in liver remains unclear. Recently apoC-II, one of the members of the apoE/C-I/C-IV/C-II gene cluster, was shown to be induced by farnesoid X receptor (FXR)/RXRα heterodimer via HCRs (11Kast H.R. Nguyen C.M. Sinal C.J. Jones S.A. Laffitte B.A. Reue K. Gonzalez F.J. Willson T.M. Edwards P.A. Mol. Endocrinol. 2001; 15: 1720-1728Crossref PubMed Scopus (224) Google Scholar). However, further study will be needed to determine whether the FXR/RXRα heterodimer also regulates the expression of other apolipoproteins (apoE, apoC-I, and apoC-IV) within the apoE/C-I/C-IV/C-II gene cluster. Members of the nuclear receptor superfamily are transcription factors that regulate gene expression through binding to specific DNA sequences known as hormone response elements (HREs). These nuclear receptors include those for steroids, thyroid hormones, vitamin D3, and retinoids as well as a large number of orphan receptors with no known ligands (12Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar). A particular member of the nuclear receptor family, the TR4 orphan nuclear receptor (TR4), is able to regulate the expression of target genes through binding to DR AGGTCA core motif sequences with variable numbers of spacer nucleotides (13Lee H.J. Lee Y. Burbach J.P. Chang C. J. Biol. Chem. 1995; 270: 30129-30133Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14Young W.J. Smith S.M. Chang C. J. Biol. Chem. 1997; 272: 3109-3116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Lee Y.F. Pan H.J. Burbach J.P. Morkin E. Chang C. J. Biol. Chem. 1997; 272: 12215-12220Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Lee Y.F. Young W.J. Burbach J.P. Chang C. J. Biol. Chem. 1998; 273: 13437-13443Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17Lee Y.F. Young W.J. Lin W.J. Shyr C.R. Chang C. J. Biol. Chem. 1999; 274: 16198-16205Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). TR4 has been shown to be highly expressed in rat primary hepatocytes (18Yan Z.H. Karam W.G. Staudinger J.L. Medvedev A. Ghanayem B.I. Jetten A.M. J. Biol. Chem. 1998; 273: 10948-10957Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To understand the physiological role of TR4, we sought to identify TR4 target genes based on the response element binding preferences of the receptor. In vitro binding assays showed that TR4 has the highest affinity for DR1 elements, and we found a DR1 site in the HCR-1, which represents a potential TR4 response element (TR4RE). We hypothesize that the apoE gene, regulated by HCR-1, is a TR4 target gene. Transcription factors and their effects on gene expression have largely been studied via in vitro binding and transfection assays in cultured cells. However, many genes have multiple response elements for different transcription factors, including nuclear receptors. Nuclear receptors have overlapping binding sites in many genes and often compete with other transcription factors for the same binding site under particular conditions (19Galson D.L. Tsuchiya T. Tendler D.S. Huang L.E. Ren Y. Ogura T. Bunn H.F. Mol. Cell. Biol. 1995; 15: 2135-2144Crossref PubMed Scopus (174) Google Scholar, 20Kassam A. Winrow C.J. Fernandez-Rachubinski F. Capone J.P. Rachubinski R.A. J. Biol. Chem. 2000; 275: 4345-4350Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Smirlis D. Muangmoonchai R. Edwards M. Phillips I.R. Shephard E.A. J. Biol. Chem. 2001; 276: 12822-12826Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Makita T. Hernandez-Hoyos G. Chen T.H. Wu H. Rothenberg E.V. Sucov H.M. Genes Dev. 2001; 15: 889-901Crossref PubMed Scopus (68) Google Scholar). In many cases, it is not easy to determine which transcription factors play major roles in vivo. Here we demonstrate that TR4 can regulate apoE expression via a DR1 element in the HCR-1 of the apoE/C-I/C-IV/C-II gene cluster and have further confirmed the role of TR4 in vivo through analysis of TR4 knockout (TR4KO) mice. Moreover, consistent with previous transgenic mouse studies showing HCR-based gene regulation, TR4 is able to modulate the expression of apoC-I and apoC-II via binding to a response element within the HCR of the apoE/C-I/C-IV/C-II gene cluster. Reagents and Plasmids—The plasmids PCMX-TR4 and pG5-luciferase (Luc) have been described previously (23Lee Y.F Shyr C.R. Thin T.H. Lin W.J. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14724-14729Crossref PubMed Scopus (83) Google Scholar), and the 11.1-kb human apoE gene was provided by Jan Breslow (Rockefeller University) (24Smith J.D. Plump A.S. Hayek T. Walsh A. Breslow J.L. J. Biol. Chem. 1990; 265: 14709-14712Abstract Full Text PDF PubMed Google Scholar). The plasmid VP16-RXRα was a gift from Dr. Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France), and pSG5-FXR and GAL4-FXR (ligand-binding domain of human FXR) were gifts from Dr. Timothy M. Willson (GlaxoSmithKline) (25Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G.T. Consler G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1857) Google Scholar). The pCMV-VP16-TR4 expression plasmid was constructed by fusion of full-length human TR4 to the transcriptional activator VP16, and GAL4-RXRα (ligand-binding domain of human RXRα) was a gift from Dr. Jaewoon Lee (POSTECH) (26Lee S.K. Anzick S.L. Choi J.-E Bubendorf L. Guan X.Y. Jung Y.K. Kallioniemi O.P. Kononen J. Trent J.M. Azorsa D. Jhun B.-H. Cheong J.H. Lee Y.C. Meltzer P.S. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). pcDNAI-HNF-4 and pCMV4-apoE4 were gifts from Dr. Margarita Hadzopoulou-Cladaras (Boston University) and Dr. Mary Jo LaDu (Evanston Northwestern Healthcare Research Institute), respectively. The apoE 5′ promoter region consisting of –1046 to +872 bp was amplified by PCR from the 11.1-kb human apoE gene, including 5.7 kb of the 5′-flanking sequence and 1.9 kb of the 3′-flanking sequence, and cloned into pGL3-Luc (Promega) to generate pGL-apoE-Luc. We first generated pGL-TK-Luc by cloning the thymidine kinase (TK) promoter (–32 to +48 bp) into pGL3-Luc (Promega). HCR-1 was amplified by PCR from HepG2 genomic DNA and subcloned into pGL-TK-Luc or pGL-apoE-Luc to generate pGL-TK-HCR-1-Luc and pGL-apoE/HCR-1-Luc, respectively. Synthesized DR1 and mutant (mt) DR1 oligonucleotides were subcloned into pGL-TK-Luc to create pGL-TK-(DR1)3-Luc and pGL-TK-mt(DR1)3-Luc, respectively. Immunohistochemistry—Two-month-old C57BL6J mice under pentobarbital anesthesia were perfused with 4% paraformaldehyde in phosphate-buffered saline. The liver was removed after adequate perfusion, and then it was further fixed in 4% paraformaldehyde in phosphate-buffered saline for 6 h. After processing and embedding in paraffin, tissue blocks were cut for staining. The liver sections were rehydrated, washed in phosphate-buffered saline, treated with 3% hydrogen peroxide in methanol for 30 min, blocked in 10% normal goat serum in phosphate-buffered saline for 30 min, and immunostained using the EnVision+ system (Dako, Carpinteria, CA). The primary antibody was a rabbit anti-TR4 polyclonal antibody (150-fold dilution). Preimmune rabbit serum (150-fold dilution) was used as a negative control in adjacent sections. After staining, the sections were developed using a 3,3′-diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA). Nuclear counterstain was performed with Gill's hematoxylin (Thermo-Shandon, Pittsburgh, PA) in all sections. Cell Culture and Transfections—HepG2 and COS-1 cells were maintained in Dulbecco's minimum essential medium containing 10% fetal calf serum. Transfections were performed by using the calcium phosphate precipitation method (14Young W.J. Smith S.M. Chang C. J. Biol. Chem. 1997; 272: 3109-3116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) or SuperFect (Qiagen, Valencia, CA). pRL-TK was used to normalize transfection efficiency in the dual luciferase reporter assay system (Promega). Metabolic Labeling of Cells and Immunoprecipitation—HepG2 cells were plated at 5 × 105 cells/60-mm dish and transfected with TR4 expression vector or empty vector (pCMX-TR4 or pCMX). After 16 h of transfection, the medium was changed, and cells were grown for another 12 h for recovery. Cells were incubated with serum- and methionine (Met)-free Dulbecco's minimum essential medium for 1 h and then pulse-labeled for 45 min with [35S]Met at 150 μCi/ml in medium. After incubation, the medium was collected, and cells were lysed by addition of 0.5 ml of lysis buffer (10 mm Na2HPO4, pH 7.5, 15 mm NaCl, 1% Nonidet P-40, 1% deoxycholate, 10 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride). Proteins newly synthesized in cell lysates and secreted into the medium were determined using trichloroacetic acid precipitation, and the required amount of each sample was aliquoted into a new tube. The volume of each sample was adjusted to 1 ml with lysis buffer, and samples were immunoprecipitated with an anti-apoE polyclonal antibody (Calbiochem). The immunoprecipitated proteins were separated by 10% SDS-PAGE, and radiolabeled apoE was quantified using a PhosphorImager (Amersham Biosciences). Western Blot Analysis—Serum samples were tested for the presence of apoE by Western blot analyses. Samples were separated by 10% SDS-PAGE. After electrophoresis, proteins were transferred from the gel to Immobilon P transfer membrane (Millipore). ApoE was resolved using an anti-apoE polyclonal antibody (Chemicon) and an alkaline phosphatase-conjugated secondary antibody (Bio-Rad), and then the relative amount of each sample was quantified using a VersaDoc imaging system (Bio-Rad). RT-PCR—Total RNA was isolated from wild-type and TR4KO mouse livers using TriZol reagent (Invitrogen), and RT-PCR was carried out using the SuperScript™ II (Invitrogen) according to the manufacturer's protocols. Briefly, after denaturation for 5 min at 65 °C in the presence of 0.5 μg of random hexamers, 3 μg of total RNA was reverse transcribed for 1 h at 43 °C with 200 units of SuperScript II in a 20-μl reaction (containing a 0.5 mm concentration of each dNTP). Two micro-liters of the cDNA sample were used as template for PCR amplification with a forward primer (5′-CAGCAGTTCATCCTAACCAGCCC-3′) specific to a region of exon 3 present in the TR4 gene in wild-type mice as well as in the targeting construct present in TR4KO mice and a reverse primer (5′-CTGCTCCGACAGCTGTAGGTC-3′) specific to a region of exon 5 replaced by the targeting construct in TR4KO mice. Hypoxanthine phosphoribosyltransferase expression was analyzed (primers: forward, 5′-GCTGGTGAAAAGGACCTCT-3′; reverse, 5′-CACAGGACTAGAACACCTGC-3′) as an internal control in the same run. Northern Blot Analysis—Total RNA was isolated using TriZol reagent (Invitrogen), and Northern blots were performed as described previously (16Lee Y.F. Young W.J. Burbach J.P. Chang C. J. Biol. Chem. 1998; 273: 13437-13443Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). An apoE probe was prepared from human apoE cDNA by AatII and DraIII digestion. Probes for mouse apoC-I (GenBank™ accession number NM_007469), apoC-II (GenBank™ accession number NM_009695), β-actin, and 18 S rRNA were generated by RT-PCR. Membrane-immobilized mRNA was hybridized with radiolabeled cDNA probes, and hybridization signals were quantified using a PhosphorImager (Amersham Biosciences). Ratios of apoE, apoC-I, and apoC-II mRNA levels relative to either 18 S rRNA or β-actin were calculated. Gel Shift Assay—Gel shift assays were performed as described previously (14Young W.J. Smith S.M. Chang C. J. Biol. Chem. 1997; 272: 3109-3116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) with the use of in vitro translated proteins or HepG2 nuclear extracts and 32P-labeled oligonucleotide probes. The following oligonucleotides were used in gel shift assays: TR4RE-DR1-apoE (5′-CTTGGGGCAGAGGTCAGAG-3′), mutated (underlined) TR4RE-DR1-apoE (5′-CTTGCCGCAGACCTCAGAG-3′), and the DR1/inverted repeat 1 (IR1) element (5′-CTTGGGGCAGAGGTCAGAGACCTCTC-3′). For the antibody supershift assays, an anti-TR4 mouse monoclonal antibody was added to the reaction. DNA-protein complexes were resolved on a 5% native gel and analyzed by PhosphorImager (Amersham Biosciences). TR4 Induces ApoE Gene Expression in Vitro—TR4 has been shown to be highly expressed in rat primary hepatocytes (18Yan Z.H. Karam W.G. Staudinger J.L. Medvedev A. Ghanayem B.I. Jetten A.M. J. Biol. Chem. 1998; 273: 10948-10957Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To verify the physiological relevance for studying the effect of TR4 on hepatic apoE expression, we performed staining of mouse liver tissue with a rabbit polyclonal antibody specific to the N-terminal domain of TR4. In mouse liver sections, the anti-TR4 antibody stained both the cytoplasm and nuclei of hepatocytes with stronger staining in the nuclei compared with the cytoplasm (Fig. 1, A and C, arrows). We also performed staining of adjacent sections with preimmune rabbit serum as a negative control to show whether this staining is TR4-specific. As shown in Fig. 1, B and D, only a weak background appears when staining with preimmune serum. These results supported our interest in further characterizing the potential regulation of hepatic apoE gene expression by TR4. We then applied a pulse labeling assay in HepG2 cells, transfected with either a TR4 expression vector or an empty vector, to study the effect of TR4 on apoE expression. After transfection and overnight recovery, cells were pulsed with [35S]Met for 45 min, and the levels of newly synthesized apoE were determined by immunoprecipitation using an anti-apoE antibody. As shown in Fig. 1E, addition of TR4 can significantly increase apoE protein expression. This induction was further confirmed at the mRNA level using Northern blot analysis. As shown in Fig. 1F, the level of apoE mRNA was higher in HepG2 cells transfected with a TR4 expression vector than in empty vector-transfected HepG2 cells. Together the data from Fig. 1 demonstrate that TR4 can induce apoE expression at both the protein and mRNA levels. Hepatic Control Region Contains a TR4 Response Element— Human hepatic control regions (HCR-1 and HCR-2) were identified in the studies of apoE/C-I/C-IV/C-II gene expression using transgenic mice (8Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar, 9Allan C.M. Walker D. Taylor J.M. J. Biol. Chem. 1995; 270: 26278-26281Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Allan C.M. Taylor S. Taylor J.M. J. Biol. Chem. 1997; 272: 29113-29119Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). HCR-1 and HCR-2 have 85% nucleotide identity and are located 18.4 and 29.4 kb downstream of the apoE transcription initiation site, respectively (Fig. 2A). In studies with transgenic mice, HCRs have been shown to have critical roles in the regulation of hepatic expression of the apoE/C-I/C-IV/C-II gene cluster. Within the entire HCR-1 774-bp region, the 319 bp at the 5′ terminus confer full HCR-1 functional activity (27Dang Q. Walker D. Taylor S. Allan C. Chin P. Fan J. Taylor J. J. Biol. Chem. 1995; 270: 22577-22585Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and sequence analysis demonstrated that this 319-bp region contains a DR1 element. Of the DR elements recognized by TR4, the receptor binds to DR1 sequences with the highest affinity (13Lee H.J. Lee Y. Burbach J.P. Chang C. J. Biol. Chem. 1995; 270: 30129-30133Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 14Young W.J. Smith S.M. Chang C. J. Biol. Chem. 1997; 272: 3109-3116Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Lee Y.F. Pan H.J. Burbach J.P. Morkin E. Chang C. J. Biol. Chem. 1997; 272: 12215-12220Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Lee Y.F. Young W.J. Burbach J.P. Chang C. J. Biol. Chem. 1998; 273: 13437-13443Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17Lee Y.F. Young W.J. Lin W.J. Shyr C.R. Chang C. J. Biol. Chem. 1999; 274: 16198-16205Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We next performed a gel shift assay to determine that this DR1 element (GGGGCAGAGGTCA named TR4RE-DR1-apoE; core motifs are underlined) functions as a TR4RE. As shown in Fig. 2B, in vitro translated TR4 protein formed a specific complex with 32P-labeled TR4RE-DR1-apoE. In contrast, the mock-translated control lysate was unable to form a complex with the DR1 element (left panel, lane 2 versus lane 1, open arrowhead). The TR4-TR4RE complex could be abolished by unlabeled TR4RE-DR1-apoE but not by mutated, unlabeled TR4RE-DR1-apoE (lane 3 versus lane 4). Moreover a supershift of the specific TR4-DR1 complex was achieved by addition of an anti-TR4 antibody (lane 5, closed arrowhead), further indicating that the TR4RE-DR1-apoE is a specific binding site for TR4. We confirmed the specific TR4-DR1 interaction when we replaced in vitro translated TR4 with HepG2 cell nuclear extracts containing endogenous TR4 (Fig. 2B, right panel). This result also confirms that TR4 protein is present in liver cells. We then used luciferase reporter assays to test whether TR4 regulates apoE gene expression through interaction with the HCR-1 region. We first linked HCR-1 to TK-luciferase (pGL-TK-HCR-1-Luc) and tested whether TR4 has any influence on the transcriptional activity of a reporter regulated by HCR-1. This construct shows high basal transcriptional activity in HepG2 cells, and co-transfection of a TR4 expression vector (pCMX-TR4) can induce luciferase activity (Fig. 2C, lane 1 versus lane 2). In contrast, the reporter construct showed very low basal transcriptional activity in COS-1 cells with significantly increased luciferase activity upon addition of the TR4 expression vector (Fig. 2C, lane 3 versus lane 4). The difference in the basal activity of the reporter in HepG2 versus COS-1 cells could be due to variation in the availability of endogenous TR4 and cooperation between endogenous TR4 and other liver-specific transcription factors to modulate HCR-1-regulated gene expression in HepG2 cells. We next addressed whether the proximal promoter of the apoE gene has any effect on HCR-1 activity. Either pGL-apoE-Luc, containing the apoE gene promoter (–1046/+872 bp) only, or pGL-apoE/HCR-1-Luc, containing the apoE gene promoter fused with the HCR-1 region, were transfected into HepG2 cells in the absence or presence of the TR4 expression vector. TR4 was found to enhance the transcriptional activity of apoE promoter and apoE promoter/HCR-1-driven luciferase reporters (Fig. 2D). However, we were unable to see any further enhancement of the TR4 effect on HCR-1 activity by addition of the apoE proximal promoter (Fig. 2, C versus D). Although TR4 stimulates apoE promoter activity, the induced promoter activity was even lower than the basal activity of pGL-apoE/HCR-1-Luc. This suggests that the apoE promoter may not have an important role in hepatic expression of the apoE gene. Indeed previous reports have indicated that the apoE promoter has no significant role in hepatic apoE expression (8Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar, 9Allan C.M. Walker D. Taylor J.M. J. Biol. Chem. 1995; 270: 26278-26281Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Allan C.M. Taylor S. Taylor J.M. J. Biol. Chem. 1997; 272: 29113-29119Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The apoE promoter may have potential TR4 binding sites as suggested by reporter gene assay even though the promoter activity does not have a significant effect on hepatic apoE expression. To define the role of TR4 relative to non-tissue-specific apoE promoter function, further study will be needed. To further confirm that TR4RE-DR1-apoE within the HCR-1 region can mediate TR4 induction of apoE expression in hepatic cells, we co-transfected a reporter with three copies of the TR4-DR1-apoE element (pGL-TK-(DR1)3-Luc). Luciferase activity was expressed based on the induction -fold relative to transfection of empty vector (pCMX, set as 1.0-fold) in each reporter gene assay. As demonstrated in Fig. 3, TR4 was able to significantly activate this pGL-TK-(DR1)3-Luc reporter in HepG2 and COS-1 cells (lane 3 versus lane 4). In contrast, TR4 only had marginal induction effects when we replaced TR4RE-DR1-apoE with mutated TR4RE-DR1-apoE (pGL-TK-(mtDR1)3-Luc) or with parent reporter plasmid (pGL-TK-Luc). Together these data strongly suggest that the DR1 element in HCR-1 is a TR4 response element important for TR4-induced transcriptional activation of the apoE gene. ApoE Expression Is Reduced in TR4KO Mouse Liver—Recently we generated TR4KO mice (in collaboration with Lexicon Genetics Inc.) by the insertion of a LacZ/Neo selection cassette between exons 4 and 5 of the TR4 gene. 2L. L. Collins, Y.-F. Lee, W.-J. Lin, C. A. Heinlein, N.-C. Liu, Y.-Ts. Chen, Y.-T. Chen, C.-R. Shyr, C. K. Meshul, H. Uno, S. M. Chou, K. A. Platt, and C. Chang, manuscript in preparation. RT-PCR analysis of total RNAs from wild-type and TR4KO mouse liver tissues confirmed the deletion of TR4 gene exons 4 and 5 in homozygous TR4KO mouse liver tissues (Fig. 4A). To determine whether TR4 could also regulate apoE expression in vivo, we examined apoE expression in wild-type (Fig. 4B, lanes 1, 3, and 5) and TR4KO mice (Fig. 4B, lanes 2, 4, and 6). From Northern blot analysis using three sets of wild-type and TR4KO mouse liver total RNAs, it was found that TR4KO apoE mRNA levels were reduced to 70% of those in wild-type mice (Fig. 4, B and D, left panel). Western blot analysis of serum apoE protein revealed that serum apoE levels of TR4KO mice were decreased by about 50% compared with wild-type serum apoE levels (Fig. 4, C and D, right panel). Considering that most apoE present in the serum is derived from the liver, the low level of serum apoE protein in TR4KO mice could be a result of the reduction of apoE mRNA expression in the liver. In vivo data collected from TR4KO mice confirm our in vitro data from HepG2 cells, which show that TR4 can induce apoE expression. Influence of Hepatocyte Nuclear Factor 4 (HNF-4) on TR4-induced ApoE Expression—Many nuclear receptors are able to bind to DR1-HRE sites, although the relative binding affinity could be influenced by the nucleotide sequences of core motifs as well as the spacer nucleotide within the particular DR1-HRE (34Nakshatri H. Bhat-Nakshatri P. Nucleic Acids Res. 1998; 26: 2491-2499Crossref PubMed Scopus (83) Google Scholar, 35Fraser J.D. Martinez V. Straney R. Briggs M.R. Nucleic Acids Res. 1998; 26: 2702-2707Crossref PubMed Scopus (56) Google Scholar). One of these nuclear receptors is HNF-4. We were interested in determining the relative influence of TR4 and HNF-4 on apoE expression. As shown in Fig. 5A, TR4 highly induced transcriptional activity of the reporter gene (pGL-TK-(DR1)3-Luc), whereas HNF-4 showed only a marginal effect on this reporter gene. To explore the mechanism of the differential induction effects of TR4 and HNF-4 on apoE expression, we performed gel shift assays. As shown in Fig. 5B, in vitro translated TR4 could strongly bind to 32P-TR4RE-DR1-apoE (lane 2, open arrowhead). However, we can only see a very weak binding complex consisting of HNF-4 and TR4RE-DR1-apoE when we replaced in vitro translated TR4 with in vitro translated HNF-4 (lane 3, arrow). Previous reports have demonstrated that changing the spacer nucleotide from A to G reduces the affinity of HNF-4 for DR1-HRE (34Nakshatri H. Bhat-Nakshatri P. Nucleic Acids Res. 1998; 26: 2491-2499Crossref PubMed Scopus (83) Google Scholar, 35Fraser J.D. Martinez V. Straney R. Briggs M.R. Nucleic Acids Res. 1998; 26: 2702-2707Crossref PubMed Scopus (56) Google Scholar). This suggests that the nucleotide sequence of TR4RE-DR1-apoE may contribute to weak binding affinity for HNF-4. The binding affinity of TR4 for TR4RE-DR1-apoE
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