A Key Role for Orphan Nuclear Receptor Liver Receptor Homologue-1 in Activation of Fatty Acid Synthase Promoter by Liver X Receptor
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m702895200
ISSN1083-351X
AutoresKaren Matsukuma, Li Wang, Mary K. Bennett, Timothy F. Osborne,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoLiver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others. Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others. Fatty acids subserve a large number of specialized cellular functions including cholesterol esterification, production of lung surfactant, mammary gland secretions, and signaling molecules. Fatty acids are also fundamental components of all biological membranes and the primary biochemical form of energy storage. Despite being a multistep enzymatic process, basic fatty acid biosynthesis in higher eukaryotes is accomplished through the catalytic activities of only two gene products, acetyl-CoA carboxylase (ACC) 3The abbreviations used are: ACC, acetyl coenzyme A carboxylase; FAS, fatty acid synthase; LXR, liver X receptor; TR, thyroid hormone receptor; RXR, retinoid X receptor; SHP, small heterodimer partner; LRH, liver receptor homologue; SREBP, sterol regulatory element-binding protein; CETP, cholesterol ester transfer protein; Q-PCR, quantitative PCR; GST, glutathioneS-transferase; siRNA, small interfering RNA; CMV, cytomegalovirus; CMX, cytomegalovirus X. and fatty acid synthase (FAS). ACC catalyzes the first step in which condensation of two acetyl-CoA molecules forms malonyl-CoA; FAS then catalyzes the remaining steps required to produce the fully saturated 16-carbon fatty acid palmitate. Although ACC is generally considered the rate-limiting enzyme in the fatty acid biosynthetic pathway, in mammals FAS appears to be independently regulated at the transcription level by a large number of nutritional, hormonal, and cellular signals, including insulin (1Wilson S.B. Back D.W. Morris Jr., S.M. Swierczynski J. Goodridge A.G. J. Biol. Chem. 1986; 261: 15179-15182Abstract Full Text PDF PubMed Google Scholar), fatty acids (2Moon Y.S. Latasa M.J. Griffin M.J. Sul H.S. J. Lipid Res. 2002; 43: 691-698Abstract Full Text Full Text PDF PubMed Google Scholar, 3Clarke S.D. Armstrong M.K. Jump D.B. J. Nutr. 1990; 120: 225-231Crossref PubMed Scopus (170) Google Scholar), thyroid hormone (4Stapleton S.R. Mitchell D.A. Salati L.M. Goodridge A.G. J. Biol. Chem. 1990; 265: 18442-18446Abstract Full Text PDF PubMed Google Scholar), sterols (5Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. 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Sul H.S. J. Biol. Chem. 1997; 272: 26367-26374Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), sterol regulatory element-binding proteins (SREBPs) (12Maganña M.M. Osborne T.F. J. Biol. Chem. 1996; 271: 32689-32694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), thyroid hormone receptor (TR) (13Xiong S. Chirala S.S. Hsu M.H. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12260-12265Crossref PubMed Scopus (36) Google Scholar), liver X receptor (LXR) (6Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar), and carbohydrate response element-binding protein (14Ishii S. Iizuka K. Miller B.C. Uyeda K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15597-15602Crossref PubMed Scopus (315) Google Scholar) as well as for general transcription factors, which play important roles in mediating the cellular response to the various signals (15Maganña M.M. Koo S.H. Towle H.C. Osborne T.F. J. Biol. Chem. 2000; 275: 4726-4733Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 16Rolland V. Liepvre X.L. Jump D.B. Lavau M. Dugail I. J. Biol. Chem. 1996; 271: 21297-21302Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17Oskouian B. Rangan V.S. Smith S. Biochem. J. 1996; 317: 257-265Crossref PubMed Scopus (9) Google Scholar). Regulation of FAS by oxysterols was reported by Joseph et al. (6Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar) who identified a DR-4-type nuclear receptor response element for the oxysterol-responsive RXR/LXR heterodimer in the FAS promoter. This DR-4 was identified in a region of the FAS promoter that was also shown to be required for TR regulation (13Xiong S. Chirala S.S. Hsu M.H. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12260-12265Crossref PubMed Scopus (36) Google Scholar). On further analysis of the FAS promoter, we identified an additional nuclear receptor half-site 21 bases downstream of the DR-4 element (Fig. 1). The region surrounding this putative nuclear receptor site did not suggest the existence of an additional DR-4-type element. However, because of its proximity to the DR-4, we hypothesized that it might play a role in the activation of FAS by either LXR or TR. In the study reported here, we show that the additional half-site binds the orphan nuclear receptor liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor and that LRH-1 binding to this site specifically augments the LXR response. In contrast, LRH-1 has no effect on thyroid hormone activation of FAS. Cell Culture—HepG2 human hepatoma cells were obtained from ATCC, cultured in Dulbecco's minimum essential medium (Invitrogen) with penicillin/streptomycin (100 μg/ml), l-glutamine (2 mm), nonessential amino acids (100 μm), sodium pyruvate (1 mm), 5 mm Hepes, pH 7.2, and 10% fetal bovine serum, and maintained at 5% CO2. One day before transfection, cells were plated at a density of 3.5 × 105 cells/well in a 6-well plate in 3 ml of normal culture medium. HEK293T human embryonic kidney cells were obtained from ATCC, cultured in Dulbecco's modified Eagle's medium high glucose (Irvine Scientific) with penicillin/streptomycin (100 μg/ml), l-glutamine (10 mm), nonessential amino acids (100 μm), 5 mm Hepes pH 7.2, and 10% fetal bovine serum, and maintained at 5% CO2. One day before transfection, cells were plated at a density of 3.5 × 105 cells/well in 6-well plates with 3 ml of normal culture medium. Transient DNA Transfections—12–24 h after plating, cells were transfected by the standard calcium phosphate method as described previously (18Yieh L. Sanchez H.B. Osborne T.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6102-6106Crossref PubMed Scopus (87) Google Scholar). Where indicated, expression vectors for CMX-LXRα (0.1 μg/well), CMX-RXRα (0.1 μg/well), CMX-TRβ (0.4 μg/well), pCI-LRH-1 (0.1–0.5 μg/well), or CMX-SHP (50 ng/well) plus individual luciferase reporters (1 μg/well) and control CMV-β-galactosidase (1 μg/well) were used. 6–8 h post-transfection, cells were glycerol-shocked for 2 min and then washed 3 times with sterile phosphate-buffered saline. Medium was replaced with defined serum-free medium (Dulbecco's minimum essential medium, penicillin/streptomycin (100 μg/ml), l-glutamine (2 mm), nonessential amino acids (100 μm), 1 mm sodium pyruvate, 5 mm Hepes, pH 7.2, insulin/transferrin/selenite (5 μg/ml; 5 μg/ml; 5 ng/ml) (Sigma), 4% bovine serum albumin (Sigma, A-3803), and 25-hydroxycholesterol (0.1 μg/ml)) plus one or more of the following: dimethyl sulfoxide (Me2SO) (0.1%) as a vehicle control, GW3965 (1 μm), LG268 (100 nm), 22-R-hydroxycholesterol (2.5 μg/ml), T0901317 (1 μm), or T3 (100 nm or 1 μm). Cells were allowed to incubate at 37 °C, 5% CO2 for an additional 36–48 h before harvesting. Plasmids—The FAS-700/+65 pGL2 luciferase reporter construct was subcloned by PCR from the rat FAS-1594/+65 pGL2 and the FAS-700/+65 MUT1 have been described previously (5Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 6Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). All other FAS mutant reporter constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene) by replacing the individual half-site sequences with an equal number of adenosine residues. The remaining reporter constructs were generated by PCR amplification and subcloning. pSynTATALuc is a reporter vector containing a minimal promoter region of the hydroxymethylglutaryl-CoA synthase promoter (–28/+39) and has been described previously (19Sanchez H.B. Yieh L. Osborne T.F. J. Biol. Chem. 1995; 270: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). The SREBP-1c promoter used here contains a sequence from –937/+29 fused to the luciferase coding sequence in pGL2 basic. The following expression vectors were provided by other laboratories: CMX-hTRβ (Barry Forman), CMX-mLXRα (Peter Tontonoz), CMX-hRXRα (Ron Evans and Bruce Blumberg), pCI-LRH-1 (Gregorio Gil), CMX-SHP (David Mangelsdorf). Enzyme Assays—At the time of harvest cells were washed once with phosphate-buffered saline and then lysed in a reporter lysis buffer (25 mm Gly-Gly, 15 mm MgSO4, 4 mm EGTA, 0.25% Triton). Luciferase activity of the lysates was measured in an Analytical Luminescence MonoLight 2010 luminometer using 20–40 μl of cell extract plus 100 μl of luciferase assay reagent (Promega) and expressed in relative light units. β-Galactosidase activity was measured by a standard colorimetric assay at 420 nm absorbance using 50–100 μl of cell lysate and 2-nitrophenyl β-galactopyranoside as the substrate. Luciferase activity for each sample was divided by the β-galactosidase activity to yield normalized relative light units. -Fold activation was determined by dividing the normalized relative light units for a given sample by the normalized relative light units for the control sample (no activators plus Me2SO). Each transfection was performed at least twice with similar results. Electrophoretic Mobility Shift Assay—In vitro transcribed and translated LXRα and RXRα proteins were generated using the T7 TnT rabbit reticulocyte lysate (Promega). One or two microliters of each translation were added to each binding mixture (containing 10 mm Hepes, pH 7.6, 50 mm NaCl, 2.5 mm MgCl2, 0.1 μg/μl poly(dI:dC), 0.05% (v/v) Nonidet P-40, 10% glycerol) in a final volume of 20 μl. For an electrophoretic mobility shift assay using GST-LRH-1, 20 ng of purified bacterially expressed GST-LRH-1 were added per reaction. A 57-base double-stranded oligo containing the wild type sequence of the three nuclear receptor half-sites was 5′ end-labeled using T4 polynucleotide kinase (U. S. Biochemical Corp.) and added to the binding mixtures (1–2 ng/reaction). Binding mixtures were incubated at 4 °C for 1–2 h. Samples were then run on 5% polyacrylamide:bisacrylamide (19:1) gels at room temperature for 1.5 h, fixed in a solution of 10% methanol, 10% acetic acid, and dried onto Whatman No. 3MM chromatography paper at 80 °C for 1 h. Dried gels were exposed to x-ray film for 12–48 h. DNA sequences (one strand from 5′-3′ only) were as follows; note that mutant bases are underlined. DNA sequences at all three half-sites were: wild type, KM 42, CTAGCACGATGACCGGTAGTAACCCCGCCTGAGGCGCCCTCCGCCAGGGTCAACGAC; mut 1, KM 44, CTAGCACGAAAAAAGGTAGTAACCCCGCCTGAGGCGCCCTCCGCCAGGGTCAACGAC; mut 2, KM 46, CTAGCACGATGACCGGTAG AAAAACCGCCTGAGGCGCCCTCCGCCAGGGTCAACGAC; mut 3, KM 48, CTAGCACGATGACCGGTAGTAACCCCGCCTGAGGCGCCCTCCGCCAAAAAAAACGAC; mut 1/2, KM 58, CTAGCACGAAAAAAGGTAGAAAAACCGCCTGAGGCGCCCTCCGCCAGGGTCAACGAC; mut 1/3, KM 56, CTAGCACGA AAAAAGGTAGTAACCCCGCCTGAGGCGCCCTCCGCCAAAAAAAACGAC; mut 2/3, KM 54, CTAGCACGATGACCGGTAG AAAAACCGCCTGAGGCGCCCTCCGCCAAAAAAAACGAC; Triple Mut, KM 70, CTAGCACGAAAAAAGGTAGAAAAACCGCCTGAGGCGCCCTCCGCCAAAAAAAACGAC. DNA sequences at the isolated third half-site were: KM 39, CTAGCGCCCTCCGCCAGGGTCAACGACCGCGCTT; MUT 3, KM 96, CTAGCGCCCTCCGCCAGTTTCAACGACCGCGCTT. Animal Studies—SHP knock-out mice described previously (20Wang 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 (396) Google Scholar) and wild type C57B1/129sv mice were maintained on a normal chow diet. Adult animals were sacrificed, and liver RNA was extracted and analyzed for FAS expression by Q-PCR as described (21Wang L. Han Y. Kim C.S. Lee Y.K. Moore D.D. J. Biol. Chem. 2003; 278: 44475-44481Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Chromatin Immunoprecipitation—B6/129 mice (6-week-old male) were purchased from Taconic and allowed to adapt for 2 weeks to a 12-h light/12-h dark cycle and sacrificed at the end of the dark cycle (8 a.m.). Livers from 4 mice were combined and placed in 40 ml of ice-cold phosphate-buffered saline containing a mixture of protease inhibitors (1 μg/ml leupeptin, 1.4 μg/ml pepstatin, and 2 μg/ml phenylmethylsulfonyl fluoride) plus 1 mm EDTA and 1 mm EGTA. The tissue was disrupted in a Tekmar Tissumizer at the lowest setting. Formaldehyde was added from a 37% stock (v/v) to a final concentration of 1%, and samples were rotated on a shaker for 6 min followed by the addition of glycine to a final concentration of 0.125 m. Samples were returned to the shaker for an additional 5 min, and then cells were collected by centrifugation (2000 rpm in Sorvall RC3B) at 4 °C. The cell pellet was washed once with homogenization buffer A (10 mm Hepes, pH 7.6), 25 mm KCl, 1 mm EDTA, 1 mm EGTA, 2 m sucrose, 10% glycerol, 0.15 mm spermine, plus protease inhibitors as above. The final pellet was resuspended in buffer A and homogenized in a Dounce homogenizer with a B pestle to release nuclei. The solution was layered over buffer A and centrifuged in a Beckman ultracentrifuge (1 h at 26,000 rpm, 4 °C), and the nuclear pellet was resuspended in nuclei lysis buffer (1% SDS, 50 mm Tris, pH 7.6, 10 mm EDTA). Nuclei were disrupted using an Ultrasonic model W-220F sonicator for 5 × 10 s to shear chromatin. Chromatin size was checked by agarose electrophoresis to ensure that the average size was between 200 and 500 bp. Aliquots were used in immunoprecipitation experiments with an antibody to LRH-1 (Santa Cruz; sc25389X) and processed for the rest of the chromatin immunoprecipitation protocol as described (22Bennett M. Toth J.I. Osborne T.F. J. Biol. Chem. 2004; 279: 37360-37367Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 23Matsukuma K.E. Bennett M.K. Huang J. Wang L. Gil G. Osborne T.F. J. Lipid Res. 2006; 47: 2754-2761Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Final DNA samples were analyzed by quantitative PCR in triplicate with a standard dilution curve of the input DNA performed in parallel. Oligo pairs for the FAS LRH-1 binding region or exon 4 from the YY1 gene used in the Q-PCR are as follows: FAS-700 (5′), ATCCTGGTCTCCAAGGTG; FAS-534 (3′), TAGGCAATAGGGTGATGGG; YY1, (5′) TCTGACGAGAGGATTGTGTGGAC; YY1, (3′) CTGAAGGGCTTTTCTCCAGTATG. LRH-1 Knockdown—HepG2 cells were plated in triplicate at 150,000 cells/well in 24-well plates in normal medium. 1–4 h after plating cells were transfected with either 5 nm siRNA molecule specific for LRH-1 (Qiagen HP GenomeWide siRNA catalog SI00056406) or a negative control siRNA sequence (Allstars negative control siRNA) using the HiPerfect transfection reagent (Qiagen). Twenty-four hours after the first transfection, cells were washed once with phosphate-buffered saline and transfected a second time in medium containing defined serum-free medium plus T0901317 (1 μm)or Me2SO (0.1%). After an additional 24 h, cells were washed once with phosphate-buffered saline and then harvested for RNA using Trizol reagent. An equal amount of RNA (0.6–1.0 μg) from each sample was used to prepare cDNAs for quantitative PCR. One microliter of each cDNA synthesis reaction was used as template for Q-PCR using the Bio-Rad iQ5 icycler and the SYBR green fluorophore. Each PCR was run in duplicate. Expression of LRH-1 and FAS was determined, and expression of each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase expression in each PCR run. PCR oligos for FAS, LRH-1, LXRα, and glyceraldehyde-3-phosphate dehydrogenase are as follows: human (hu) FAS, (5′), AACTCCAAGGACACAGTCACCAT; hu FAS (3′), CAGCTGCTCCACGAACTCAA; hu LRH-1 (5′), CTGATACTGGAACTTTTGAA; hu LRH-1 (3′), CTTCATTTGGTCATCAACCTT; hu LXRα (5′), GGAGGTACAACCCTGGGAGT; hu LXRα (3′), AGCAATGAGCAAGGCAAACT; hu glyceral-dehyde-3-phosphate dehydrogenase (5′), GAAGGTGAAGGTCGGAGTC; hu glyceraldehyde-3-phosphate dehydrogenase (3′), GAAGATGGTGATGGGATTTC. In our analysis of the FAS promoter, we noted a highly conserved sequence 21 bases downstream of the RXR/LXR response element bearing a strong resemblance to other nuclear receptor half-sites (Fig. 1). To determine whether this sequence played a role in the FAS LXR response, we first compared LXR activation of the wild type FAS promoter to LXR activation of similar reporter constructs containing mutations in each of the three half-sites (Fig. 2A). As expected, mutations of either half-site 1 or 2 (comprising the known LXR DR-4 element) resulted in a significant decrease in LXR activation. Surprisingly, however, mutation of the third half-site alone also resulted in a substantial defect in LXR activation, suggesting that the third half-site played an important and previously unappreciated role in LXR activation of FAS. Furthermore, mutation of all three half-sites simultaneously resulted in the most significant defect. Even with all three sites mutated there was still a 6-fold activation by LXR/RXR. This residual activity is due to LXR induction of SREBP-1c, which subsequently activates the FAS promoter through binding to sites in the proximal promoter (6Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). To better understand the mechanism through which the third half-site exerts its effect on LXR activation of FAS, we examined binding of in vitro translated RXRα and LXRα proteins to a 32P-labeled DNA probe containing all three nuclear receptor half-sites (Fig. 2B). The addition of both RXRα and LXRα proteins resulted in a single band (lane 1) that migrated in parallel with a known RXRα/LXRα heterodimer on a consensus DR-4. The addition of a 200-fold excess of unlabeled wild type competitor DNA effectively competed for RXRα/LXRα heterodimer binding (lane 2); however, competitor DNAs containing mutations in the first and/or second half-sites (lanes 3, 4, 6–9) were unable to compete significantly for heterodimer binding. In contrast, competitor DNA containing a mutation in the third half-site alone effectively competed for RXRα/LXRα heterodimer binding (lane 5). Thus, the third half-site did not play a significant role in RXRα/LXRα binding to this region of the promoter despite being an important determinant of the FAS LXR response. Two other known LXR target genes, murine CYP7A1 (24Lu 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 (1228) Google Scholar) and human CETP (25Luo Y. Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 24767-24773Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), require binding of the monomeric nuclear receptor LRH-1 (also known as fetoprotein transcription factor and CYP7A1 promoter binding factor) at a site proximal the LXR element, for efficient LXR activation. To determine whether LRH-1 could bind to the FAS third half-site and potentiate the LXR response of the FAS gene, we first assessed binding of recombinant LRH-1 protein to the FAS third half-site by electrophoretic mobility shift assay (Fig. 3A). Incubation of 20 ng of GST-LRH-1 with the labeled third half-site resulted in a single shifted band (lane 2) that migrated in parallel with GST-LRH-1 bound to a known LRH-1 binding site from the CYP8B1 promoter (lane 10). The addition of a 50- and 200-fold excess of unlabeled competitor DNA containing the wild type third half-site sequence effectively competed for GST-LRH-1 binding to the FAS half-site in a dose-dependent manner (lanes 3 and 4) as did competitor DNA containing the LRH-1 binding site from the CYP8B1 promoter (lanes 7 and 8). In contrast, the addition of competitor DNA containing a mutation in the third half-site did not compete for LRH-1 binding (lanes 5 and 6). We next investigated direct binding of LRH-1 to the endogenous FAS promoter by chromatin immunoprecipitation in normal mouse liver using an antibody to LRH-1 and oligonucleotides that amplify the relevant region of the FAS promoter (Fig. 3B). Here, there was significant enrichment of the FAS promoter by the LRH-1 antibody relative to an IgG control, and this effect was specific as there was no enrichment of DNA from the YY1 locus analyzed in parallel. To determine whether LRH-1 might activate the FAS promoter and augment the LXR response, we turned to HEK293T cells because HepG2 cells contain a significant level of endogenous LRH-1. To eliminate any indirect effects mediated through LXR activation of SREBP-1c (an LXR target gene and direct activator of FAS) (Fig. 2 and Refs. 5Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar and 6Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar), a truncated wild type FAS promoter construct was constructed that contained the three nuclear receptor half-sites but lacked the downstream SREBP recognition motifs (FAS-700/-574) fused to a minimal TATA box containing promoter. This was transfected into HEK293T human embryonic kidney cells with an expression vector for LRH-1 along with the FAS promoter construct containing the three nuclear receptor half-sites (Fig. 4A). Because the RXR/LXR heterodimer is permissive and, thus, can be activated simultaneously by an RXR ligand and an LXR ligand (26Willy P.J. Mangelsdorf D.J. Genes Dev. 1997; 11: 289-298Crossref PubMed Scopus (141) Google Scholar), we compared activation of the FAS promoter by a synthetic RXR agonist (LG268), an oxysterol known to activate LXR (22-R-hydroxycholesterol) or both in combination. In the absence of transfected LRH-1, the addition of each ligand or the combination of both resulted in 2–5-fold increases in FAS activation, as is consistent with the FAS being an RXR/LXR target gene. In the presence of transfected LRH-1, however, this modest response increased to 20.5-fold, whereas LXR ligands or LRH-1 alone activated FAS 4.4- and 2.9-fold, respectively. Furthermore, the effect of LRH-1 on the LXR response was specific to FAS, as no such effect was noted on a control promoter construct analyzed in parallel. To better understand the contribution of each half-site to the LRH-1 potentiation effect, we used mutant versions of this FAS reporter construct containing either a double mutation of both halves of the DR-4 or a mutation in the putative LRH-1 (third) half-site (Fig. 4B). As in the previous experiment, LXR activation of the corresponding wild type FAS promoter was dramatically increased in the presence of LRH-1. Furthermore, the addition of an expression vector for SHP, a novel non-DNA binding nuclear receptor known to be a potent inhibitor of LRH-1 activity in other promoters, significantly blunted this response. The FAS promoter construct containing a mutation in the DR-4 (MUT1/2) was defective for RXR/LXR signaling irrespective of the presence of LRH-1. Importantly, the magnitude of activation by LRH-1 alone on this mutant construct was similar to that seen on the wild type promoter, demonstrating an intact LRH-1 response. Activation of the promoter construct containing a mutation in the third half-site (MUT 3) by RXR/LXR alone was comparable with that seen on the wild type construct; however, activation by LRH-1 alone was lost as was the concerted activation by both LXR and LRH-1. The residual inhibitory effect of SHP in the absence of the LRH-1 site was likely due to its known ability to inhibit LXR activation (27Brendel C. Schoonjans K. Botrugno O.A. Treuter E. Auwerx J. Mol. Endocrinol. 2002; 16: 2065-2076Crossref PubMed Scopus (166) Google Scholar). Thus, SHP likely inhibits FAS gene expression through interactions with both LRH-1 and LXR. We next used an siRNA approach to evaluate the effects of specifically reducing endogenous LRH-1 expression on the LXR-mediated induction of FAS in HepG2 cells (Fig. 5A). We used HepG2 cells because an siRNA approach has been used previously to silence LRH-1 gene expression (28Venteclef N. Smith J.C. Goodwin B. Delerive P. Mol. Cell. Biol. 2006; 26: 6799-6807Crossref PubMed Scopus (43) Google Scholar). The siRNA specifically reduced endogenous LRH-1 mRNA by 30–50%, which resulted in a significant blunting of the response of FAS to LXR activation. To rule out off-target effects of the siRNA, we also measured mRNA expression of LXRα in these cells. As expected, the addition of the siRNA did not decrease LXRα levels. Although LRH-1 knock-out mice die early in embryogenesis and are, thus, unavailable for study (29Pare J.F. Malenfant D. Courtemanche C. Jacob-Wagner M. Roy S. Allard D. Belanger L. J. Biol. Chem. 2004; 279: 21206-21216Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), SHP knock-out mice are viable (20Wang 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 (396) Google Scholar). Given that SHP is a potent inhibitor of LRH-1 (24Lu 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 (1228) Google Scholar, 30Datta S. Wang L. Moore D.D. Osborne T.F. J. Biol. Chem. 2006; 281: 807-812Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 31Goodwin 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 (1513) Google Scholar), it follows that if FAS is indeed an LRH-1 target gene, FAS mRNA expression would be elevated in SHP knock-out mice. Consistent with this hypothesis, our animal studies showed that FAS mRNA expression in SHP knockout mice was 6.8-fold higher than in matched wild type controls (Fig. 5B). In a previous report LRH-1 was shown to augment the LXR response of SREBP-1c (43Watanabe M. Houten S.M. Wang L. Moschetta A. Mangelsdorf D.J. Heyman R.A. Moore D.D. Auwerx J. J. Clin. Investig. 2004; 113: 1408-1418Crossref PubMed Scopus (983) Google Scholar). However, despite localization of the effect to the proximal 300 bases of the SREBP-1c promoter, an LRH-1 site could not be identified, and
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