Peroxisome Proliferator-activated Receptor γ Stimulation of Adipocyte ApoE Gene Transcription Mediated by the Liver Receptor X Pathway
2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês
10.1074/jbc.m808482200
ISSN1083-351X
Autores Tópico(s)Lipid metabolism and biosynthesis
ResumoPeroxisome proliferator-activated receptor (PPARγ) agonists increase insulin sensitivity in humans and are useful for treating human diabetes. Treatment with these agonists leads to increased apoE expression and triglyceride accumulation in adipocytes. The importance of apoE for adipocyte triglyceride accumulation is demonstrated by observations that triglyceride accumulation is impaired in apoE knockout adipocytes treated with PPARγ agonists. The current studies investigate the molecular mechanism for PPARγ stimulation of the adipocyte apoE gene and demonstrate that the liver receptor X (LXR) response element within an apoE gene downstream enhancer is required for the apoE response to PPARγ agonists. The response of the apoE gene to treatment with PPARγ agonists was delayed beyond 12 h suggesting the involvement of an intermediary pathway. The combined addition of PPARγ and LXR agonists did not increase apoE response beyond that observed with addition of either alone. Deletion or mutation of the LXR response element completely eliminated the adipocyte apoE gene response to a PPARγ agonist. Chromatin immunoprecipitation analyses performed using isolated adipocytes, or adipose tissue from mice treated with PPARγ agonists, showed increased LXR binding to the apoE gene after PPARγ agonist treatment. Knockdown of LXR expression completely eliminated the increase in apoE message, protein, and triglyceride in response to PPARγ stimulation. The LXR response element has been previously shown to mediate sterol responsiveness of the apoE gene, and apoE expression plays an important role in adipocyte triglyceride balance. The current observations suggest that the PPARγ-LXR-apoE regulatory cascade could be an important molecular link for cross-talk between adipocyte triglyceride and cholesterol homeostasis. Peroxisome proliferator-activated receptor (PPARγ) agonists increase insulin sensitivity in humans and are useful for treating human diabetes. Treatment with these agonists leads to increased apoE expression and triglyceride accumulation in adipocytes. The importance of apoE for adipocyte triglyceride accumulation is demonstrated by observations that triglyceride accumulation is impaired in apoE knockout adipocytes treated with PPARγ agonists. The current studies investigate the molecular mechanism for PPARγ stimulation of the adipocyte apoE gene and demonstrate that the liver receptor X (LXR) response element within an apoE gene downstream enhancer is required for the apoE response to PPARγ agonists. The response of the apoE gene to treatment with PPARγ agonists was delayed beyond 12 h suggesting the involvement of an intermediary pathway. The combined addition of PPARγ and LXR agonists did not increase apoE response beyond that observed with addition of either alone. Deletion or mutation of the LXR response element completely eliminated the adipocyte apoE gene response to a PPARγ agonist. Chromatin immunoprecipitation analyses performed using isolated adipocytes, or adipose tissue from mice treated with PPARγ agonists, showed increased LXR binding to the apoE gene after PPARγ agonist treatment. Knockdown of LXR expression completely eliminated the increase in apoE message, protein, and triglyceride in response to PPARγ stimulation. The LXR response element has been previously shown to mediate sterol responsiveness of the apoE gene, and apoE expression plays an important role in adipocyte triglyceride balance. The current observations suggest that the PPARγ-LXR-apoE regulatory cascade could be an important molecular link for cross-talk between adipocyte triglyceride and cholesterol homeostasis. PPARγ 2The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; TGD, triglyceride droplet; TZD, thiazolidinedione; TG, triglyceride; LXR, liver receptor X; LXRE, LXR enhancer; 22-OHch, 22-hydroxycholesterol; EKO, apoE knockout; WT, wild type; RT, reverse transcription; siRNA, small interference RNA; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ME, macrophage enhancer. is highly expressed in adipocytes where it modulates the expression of a program of genes involved in adipocyte function (1Tontonoz P. Spiegelman B.M. Annu. Rev. Biochem... 2008; 77: 289-312Google Scholar). These include genes involved in TGD and fatty acid metabolism and in the expression of adipokines. Adipocyte lipid metabolism and the secretion of adipokines have a powerful effect on systemic substrate utilization and on overall organismal energy metabolism (2Scherer P.E. Diabetes.. 2006; 55: 1537-1545Google Scholar, 3Fantuzzi G. Mazzone T. Arterioscler. Thromb. Vasc. Biol... 2007; 27: 996-1003Google Scholar). TZD drugs, currently in clinical use for treatment of human diabetes, are established ligands of adipocyte PPARγ receptors, and we have previously shown that adipocyte apoE expression is responsive to treatment with these drugs (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar). Zechner and colleagues first described expression of apoE by adipocytes in 1991 (5Zechner R. Moser R. Newman T.C. Fried S.K. Breslow J.L. J. Biol. Chem... 1991; 266: 10583-10588Google Scholar), but only recently has additional information been provided regarding regulation and function of adipocyte apoE (6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar, 7Yue L. Christman J.W. Mazzone T. Endocrinology.. 2008; 149: 4051-4058Google Scholar, 8Espiritu D.J. Mazzone T. Diabetes.. 2008; 57: 2992-2998Google Scholar, 9Huang Z.H. Luque R.M. Kineman R.D. Mazzone T. Am. J. Physiol... 2007; 293: E203-E209Google Scholar-10Rao P. Huang Z.H. Mazzone T. J. Clin. Endocrinol. Metab... 2007; 92: 4366-4372Google Scholar). Treating isolated adipocytes or intact humans with PPARγ agonists increases adipocyte and adipose tissue apoE gene expression (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar, 6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar). The pro-inflammatory cytokine, tumor necrosis factor-α, reduces adipocyte apoE expression, an effect mediated by the NF-κB transcriptional complex binding an element in the apoE gene proximal promoter (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar, 7Yue L. Christman J.W. Mazzone T. Endocrinology.. 2008; 149: 4051-4058Google Scholar). Reactive oxygen species produced by the stromovascular fraction of adipose tissue harvested from obese mice also reduce adipocyte apoE expression via the NF-κB pathway (8Espiritu D.J. Mazzone T. Diabetes.. 2008; 57: 2992-2998Google Scholar). With respect to apoE function, adipocytes from EKO mice are smaller than adipocytes from WT mice, and cultured EKO adipocytes and adipose tissue accumulate less TG compared with WT cells after incubation with apoE-containing lipoproteins (6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar). This defect in TG accumulation can be corrected by the adenoviral expression of apoE in EKO adipocytes. Furthermore, in EKO adipocytes, TG accumulation in response to treatment with PPARγ agonists is impaired compared with WT adipocytes (6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar). All of these observations support a role for apoE in adipocyte TG metabolism. Such a role for apoE is different than what has been described for other cell types, for example macrophages, in which apoE has been shown to primarily play a role in cellular sterol metabolism (11Mazzone T. Reardon C. J. Lipid Res... 1994; 35: 1345-1353Google Scholar, 12Lin C.-Y. Duan H. Mazzone T. J. Lipid Res... 1999; 40: 1618-1626Google Scholar). Its role in these cells includes modulating sterol efflux and/or the subcellular distribution of sterol. Consistent with its role in cellular sterol metabolism, the apoE gene is strongly regulated by cellular sterol content acting via the sterol-sensing LXR pathway and LXR response elements present in two duplicated downstream apoE gene enhancers, ME.1 and ME.2 (13Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kas H.R. Mangeldorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 507-512Google Scholar).In previous studies, we established that the response of the apoE gene to PPARγ agonists was not mediated by the apoE proximal promoter but required the presence of at least one of these two apoE downstream gene enhancers (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar). Based on the requirement for a downstream enhancer, the presence of an LXRE in the enhancer, the absence of a clearly identifiable PPRE within this enhancer, and the recently established positive effect of PPARγ agonists on LXR gene transcription (14Chawla A. Boisvert W.A. Lee C.H. Laffitte B.A. Barak Y. Joseph S.B. Liao D. Nagy L. Edwards P.A. Curtiss L.K. Evans R.M. Tontonoz P. Mol. Cell.. 2001; 7: 161-171Google Scholar), we hypothesized that the response of the adipocyte apoE gene to PPARγ agonists could be mediated by the LXR pathway (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar). Based on similar considerations, a role for the LXR pathway has been posited as mediating the effect of PPARγ agonists on ABCA1 expression in macrophages (14Chawla A. Boisvert W.A. Lee C.H. Laffitte B.A. Barak Y. Joseph S.B. Liao D. Nagy L. Edwards P.A. Curtiss L.K. Evans R.M. Tontonoz P. Mol. Cell.. 2001; 7: 161-171Google Scholar). Because of observations supporting a role for apoE in adipocyte TG metabolism, the involvement of the LXR pathway for mediating the PPARγ response of the adipocyte apoE gene could provide an important nexus of cross-talk between adipocyte TG and sterol metabolism. The studies reported in this manuscript examine the molecular mechanism for PPARγ stimulation of adipocyte apoE gene transcription and establish an important role for the LXR pathway for mediating this response.EXPERIMENTAL PROCEDURESMaterials—Cell culture media, supplements, and antibiotics were purchased from Invitrogen. Insulin, 1-methyl-3-isobutylxanthine, dexamethasone, GW3965, and 22-OHch were obtained from Sigma. Ciglitazone and rosiglitazone were purchased from Cayman Chemical (Ann Arbor, MI). Pioglitazone was obtained from Takeda Pharmaceutical Co. Antibodies to LXRα/β were obtained from Santa Cruz Biotechnology.Animals—Eight-week-old C57BL/6J mice were purchased from Jackson Laboratories. Mice were housed in a temperature-controlled room with a 12-h light/dark cycle and were given free access to standard chow and water for 10 days. Pioglitazone (25 mg/kg/day) mixed with chow or the control chow diet was fed for an additional 5 days before mice were sacrificed for isolation of adipose tissue. Intra-abdominal white adipose tissue was collected in ice-cold phosphate-buffered saline. For real-time PCR quantitation of apoE mRNA abundance, total RNA was extracted for synthesis of first-strand cDNA. For the in vivo chromatin immunoprecipitation assay, adipose tissue was prepared according to the EpiQuik™ tissue chromatin immunoprecipitation kit (Epigentek, Brooklyn, NY).Cell Culture—3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained for passage in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at <70% confluence. For experiments, differentiation was induced at 2 days post confluence by incubation for 3 days in culture medium containing 0.25 μm dexamethasone, 0.5 mm 3-isobutyl-l-methylxanthine, and 10 μg/ml insulin. This was followed by 10% fetal bovine serum/Dulbecco's modified Eagle's medium containing 5 μg/ml insulin that was renewed every 2 or 3 days. Experiments were performed on post differentiation days 8–10. Triglyceride content of cells was measured using a kit from Wako Chemicals as previously described (6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar).Real-time RT-PCR and Immunoblot Analysis—Total RNA was extracted from cells or tissues after the treatments described in figure legends. Each sample of first strand cDNA was synthesized from 1 μg of total RNA using a Thermo-Script™ RT-PCR system according to the manufacturer's instructions (Invitrogen). cDNA (1.5 μl) was amplified by real-time PCR using a TaqSYBR green supermix with ROX (Bio-Rad). Normalization for loading was accomplished by assessing β-actin message abundance. Individual samples were analyzed in triplicate using an Mx3000P quantitative PCR system (Stratagene). Levels for RNA are reported as -fold change compared with control using the comparative quantitation analysis software available on the Mx3000P. Product purity for PCR reactions was confirmed by examination melting curves for the presence of a single peak. Immunoblots on cells or tissues were performed, and the results were quantitated as previously described (7Yue L. Christman J.W. Mazzone T. Endocrinology.. 2008; 149: 4051-4058Google Scholar).Site-directed Mutagenesis—The human apoE promoter fragment –503/+86 alone or with the human apoE ME2 downstream enhancer (7Yue L. Christman J.W. Mazzone T. Endocrinology.. 2008; 149: 4051-4058Google Scholar, 13Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kas H.R. Mangeldorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 507-512Google Scholar) were cloned into pGL3 luciferase vector as previously described to give rise to pGL503 or pGL503en, respectively. Site-directed deletion or mutation was performed using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The primer with deleted nucleotides (indicated by the dashed line) was: 5′-GGA CAC CCT CCC CAT CAG CTG CCA————–AAG GCA GCT GGT GGG GAA GGC ATT GG-3′. The primer with mutated nucleotides (as indicated in small cap letters) was: 5′-CCC CAT CAG CTG CCA Gaa TCA CTG GCa aTC AAA GGC AGC TGG TG-3′. These primers and their complementary sequences were used to disrupt the LXR response element in ME2. All deletions and mutations were confirmed by DNA sequencing.Transient Transfection and Luciferase Activity Assay—Transient transfection of luciferase reporter plasmids was performed using Lipofectamine 2000 (Invitrogen) according to the protocol recommended by the manufacturer in the 12-well plate format. Luciferase assays were performed using the luciferase assay system (Promega, Madison, WI). Following the treatments described in the figure legends, cells were washed twice in cold phosphate-buffered saline and lysed in 120 μl of cell culture lysis reagent provided in the luciferase assay kit. 10 μl of the cell lysate was mixed with 50 μl of luciferase assay solution, and luminescence was determined using a Turner Design Luminometer. All experiments were performed in triplicate and normalized to the activity produced by a cotransfected β-galactosidase expression vector. For RNA interference, differentiated 3T3-L1 cells were trypsinized and plated in fresh growth medium (3 × 105 per ml) and transferred into 12-well plates at 1 ml per well. In each well, 100 pm of a mixture of siRNA targeted to LXRα and LXRβ (L-040649-01-0005, Mouse NR1H3, NM_013839 and L-042839-00-0005, Mouse NR1H2, NM_009473, respectively) complexed with 2 ml of Lipofectamine was added. A non-targeting siRNA provided by the manufacturer was used as a control.Chromatin Immunoprecipitation—3T3-L1 adipocytes were treated as described in the figure legends. Chromatin immunoprecipitation was carried out using the ChIP-IT Express kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions as previously described (7Yue L. Christman J.W. Mazzone T. Endocrinology.. 2008; 149: 4051-4058Google Scholar). Briefly, the cells were fixed, and the isolated nuclear fraction was digested for 13 min at 37 °C by an enzymatic shearing mixture. The digestate was visualized on an agarose gel to ensure shearing efficiency resulted in DNA fragments ranging from 200 to 500 bp. After centrifugation, 10 μl of the supernatant containing sheared chromatin was used as the assay input. The sheared chromatin/ protein was immunoprecipitated using an anti-LXRα/β antibody (sc-13068, Santa Cruz Biotechnology) at 4 °C overnight. After immunoprecipitation, samples were centrifuged, washed, and reverse cross-linked prior to being treated with ribonuclease A and proteinase K. The DNA was purified with DNA mini columns provided with the kit, and real-time PCR was performed with primers (A 5′-AAC TGA CAC GTG GGT TGC AGG GGC AAC A-3′) and the reverse primer (5′-CAA CAT CCT TTC TCT CAG GCT AGA GTC C-3′), designed to amplify a 152-bp portion of the apoE enhancer containing the LXRE. As an internal control, an RNA polymerase II antibody (sc-899, Santa Cruz Biotechnology) was used to immunoprecipitate the mouse GAPDH promoter.The in vivo ChIP was performed with freshly isolated adipose tissue using the EpiQuik™ tissue ChIP kit according to the manufacturer's instructions. 400 mg of adipose tissue in 2 ml of homogenizing buffer was homogenized using 20 strokes of a Dounce homogenizer. The homogenate was centrifuged at 5000 rpm for 10 min at 4 °C, and the pellet was re-suspended in 1 ml of CP3 buffer (included with the kit) with protease inhibitors. The DNA was sheared by sonication with 10 pulses of 15 s each at 25% Power using a Vibracell 3-mm microtip probe with 40-s incubation on ice between each pulse. The sheared DNA was used for immunoprecipitation as described above.Statistical Analysis—All the data are presented at means ± S.D. values. Statistical differences were evaluated using analysis of variance (SPSS, Chicago, IL). p < 0.05 was considered significant.RESULTSFig. 1 presents the results of detailed dose response and time course analyses for the response of apoE gene to treatment with rosiglitazone. The dose-related increase with rosiglitazone establishes the apoE response after treatment of adipocytes with three different PPARγ agonists, rosiglitazone (Fig. 1A), ciglitazone, and pioglitazone (4Yue L. Rassouli N. Ranganathan G. Kern P.A. Mazzone T. J. Biol. Chem... 2004; 279: 47626-47632Google Scholar, 6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar). The time course for the response of the apoE gene to treatment with rosiglitazone (Fig. 1B) demonstrates that the first measurable increase in apoE mRNA levels after addition of rosiglitazone is not detected until after 12 h. This somewhat prolonged lag time for the apoE gene response is consistent with the hypothesis that the apoE gene does not respond directly to PPARγ agonists and that the response requires involvement of an intermediary pathway. Based on considerations presented in the introduction, we focused on a potential intermediary role for the LXR pathway. The LXR gene has been shown to be a target of PPARγ agonists (14Chawla A. Boisvert W.A. Lee C.H. Laffitte B.A. Barak Y. Joseph S.B. Liao D. Nagy L. Edwards P.A. Curtiss L.K. Evans R.M. Tontonoz P. Mol. Cell.. 2001; 7: 161-171Google Scholar, 15Juvet L.K. Andresen S.M. Schuster G.U. Dalen K.T. Tobin K.A. Hollung K. Haugen F. Jacinto S. Ulven S.M. Bamberg K. Gustafsson J.-A. Nebb H.I. Mol. Endocrinol... 2008; 17: 172-182Google Scholar), and we examined the response of LXR protein levels in our model system. Fig. 1 (C and D) shows that treatment of 3T3-L1 adipocytes with rosiglitazone increased total cellular (C) and nuclear (D) LXR protein abundance.We next measured the response of the adipocyte apoE gene, and of an apoE gene reporter construct, to incubation with an LXR agonist, a PPARγ agonist, or a combination of these (Fig. 2). The addition of 22-OHch (an oxysterol LXR agonist) alone, or ciglitazone alone, led to a 2- to 3-fold increase in the expression of the adipocyte apoE gene and of a reporter construct containing 503 bp of the proximal apoE gene promoter and the apoE gene enhancer ME2 (Fig. 2, A and B). Addition of the agents together, however, did not produce a greater increase in apoE or reporter gene response compared with either agent alone. The reporter construct without ME2 did not respond to either 22-OHch or ciglitazone. An experiment similar to that shown in Fig. 2A, but conducted with the alternate agonists, rosiglitazone and GW3965, gave similar results. Either agent alone significantly stimulated apoE mRNA abundance, but the effect of the agents together was not additive. The absence of an additive response to the combined addition of PPARγ and LXR agonists provided further support for the hypothesis that these agonists act via a common pathway to stimulate adipocyte apoE gene expression.FIGURE 2Activation of the adipocyte apoE gene by PPARγ and LXR ligands is not additive. A, 3T3-L1 cells were treated with ethanol vehicle, 10 μm 22-OHch, 20 μm ciglitazone (Cig), or a combination of these for 48 h before harvest to measure apoE mRNA level. B, 3T3-L1 adipocytes were transfected with the indicated apoE reporter gene constructs and treated as described in A. Luciferase activity was measured as described under "Experimental Procedures." C, cells were treated with 10 μm rosiglitazone or 1 μm GW3965 for 48 h and harvested as described in A. *, p < 0.005 for comparison to untreated control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The quantitatively similar response of the endogenous apoE gene and the pGL503en reporter construct to ciglitazone in Fig. 2 indicated that the important gene elements required for increased apoE gene expression in response to PPARγ agonists were contained within this reporter construct. Using the results in Figs. 1 and 2 as a foundation, we designed experiments to more definitively examine the role of the LXR pathway in mediating the PPARγ response of the apoE gene by (a) evaluating the need for the apoE gene LXRE, (b) measuring the recruitment of LXR to the apoE gene in response to PPARγ agonist stimulation in vitro and in vivo, and (c) evaluating the effect of silencing LXR protein expression. Fig. 3 (A and B) provides the details of the four reporter gene constructs used to examine the importance of the apoE gene LXRE. These constructs include a proximal apoE gene promoter construct without the ME2 downstream enhancer, the proximal promoter construct with ME2, the enhancer construct in which the LXR response element is deleted, and the enhancer construct in which the LXR element is mutated. The specific nucleotides deleted or mutated are shown in Fig. 3B. Fig. 3C shows that the promoter construct without ME2 failed to respond to ciglitazone, whereas the promoter construct containing ME2 responded with an approximate 3-fold increase to treatment with ciglitazone. Deletion of the LXR response element within ME2 completely eliminated the response to ciglitazone. Fig. 3D shows that the pGL503en construct responded with 2- to 3-fold increased expression after treatment with 22-OHch or ciglitazone. Mutation of four nucleotides within the LXR element, however, completely eliminated the reporter gene response to both agonists.FIGURE 3Stimulation of an apoE gene reporter construct by ciglitazone (Cig) requires an intact LXRE. A, the reporter constructs used to evaluate apoE gene elements responsive to PPARγ agonists are shown. The numbers on each side of the solid bars indicate the position in base pairs relative to transcription initiation site. The solid bars represent exon-1 of the apoE gene. The open box represents the apoE ME2 enhancer element. The presence of an intact (en), deleted (en. del), or mutated (en. mut) LXRE is indicated. B, sequences of the native apoE gene LXRE, the mutated LXRE, and the LXRE deletion. Lowercase letters indicate mutated nucleotides. Hatch marks represent deleted nucleotides. C, 3T3-L1 cells were transfected with the indicated constructs and incubated with or without 20 μm cig for 48 h. Normalized luciferase activity relative to untreated control is shown. D, 3T3-L1 cells were transfected with the indicated constructs and treated with 20 μm cig or 10 μm 22-OHch for 48 h as shown. Normalized luciferase activity relative to untreated control is shown. *, p < 0.001 for comparison to untreated control. Results shown are mean ± S.D. of three independent experiments each done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figs. 4 and 5 show the results of ChIP analyses and confirm that treatment of intact cells (Fig. 4) or mice (Fig. 5) with PPARγ agonists leads to increased LXR binding to the apoE gene. Fig. 4A shows that treatment of intact adipocytes with ciglitazone or 22-OHch increased LXR binding to the apoE gene. A representative agarose gel shows the binding of RNA polymerase II to the GAPDH gene (as an internal control) and the binding of LXR to the apoE gene in response to treatment of adipocytes with 22-OHch or ciglitazone. The bar figure represents PCR quantitation of three separate ChIP analyses, all corrected for the internal GAPDH control, and shows a 4-fold increase in LXR binding to the apoE gene after treatment of adipocytes with ciglitazone. Fig. 4B shows a similar result from an identical experiment using a different PPARγ agonist, rosiglitazone. For Fig. 5, mice were treated for 5 days with pioglitazone at 25 mg/kg/day. At the end of that time, freshly isolated adipose tissue demonstrated a 3-fold increase in apoE mRNA abundance (Fig. 5A). ChIP analysis using freshly isolated adipose tissue demonstrated significantly more LXR protein binding (3.7-fold) at the apoE gene enhancer element.FIGURE 4ChIP analysis of LXR binding to the apoE gene in response to the treatment with PPARγ ligands. 3T3-L1 cells were treated with 10 μg 22-OHch or ciglitazone (Cig)(A) or rosiglitazone (Rosi)(B) for 48 h and then harvested for ChIP analysis as described under "Experimental Procedures." After immunoprecipitation, a 152-bp amplicon of the apoE enhancer containing the LXRE was amplified by PCR. The amount of LXR bound to the LXR enhancer was assessed using real-time PCR to quantitate immunoisolated DNA. Representative images of agarose gels show RNA polymerase II binding to the GAPDH promoter (as an internal control) and LXR binding to apoE. The real-time PCR results are mean ± S.D. from three different ChIP analyses, each done in triplicate. *, p < 0.05 compared with untreated control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Treatment with PPARγ ligand in vivo activates LXR binding to the apoE gene. A, mice (three per group) were fed chow or chow plus pioglitazone (Pio, 25 mg/kg per day) for 5 days before harvest of adipose tissue. A, total RNA was extracted, and apoE mRNA was quantitated by RT-PCR as described under "Experimental Procedures." *, p < 0.005 compared with untreated controls. B, chromatin immunoprecipitation assay was performed as described under "Experimental Procedures" using freshly isolated adipose tissue from pioglitazone-treated and untreated mice. A representative agarose gel shows RNA polymerase II binding to the GAPDH gene (as an internal control) and LXR binding to the apoE gene. Real-time PCR quantitation of immunoisolated DNA after LXR immunoprecipitation is shown as -fold change in treated compared with control mice. *, p < 0.005 compared with untreated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The above results established that treatment with PPARγ agonists increased LXR binding to the apoE gene enhancer in vitro and in vivo, and that the LXR response element in the apoE gene enhancer was required for increased apoE gene expression after PPARγ stimulation. We next performed experiments to further confirm that an intact adipocyte LXR pathway was required for the response of the endogenous adipocyte apoE gene to PPARγ stimulation. Cells were transfected with an siRNA for LXR, or a control siRNA, and after 24 h they were treated with PPARγ agonists as indicated (Fig. 6). The results in Fig. 6 (A and B) show that treatment with a combined LXRα/LXRβ siRNA leads to a 65–80% reduction in LXRα (A) or LXRβ (B) mRNA in unstimulated adipocytes. Further, treatment with the LXR siRNA completely eliminated the response to ciglitazone that is easily observed in cells treated with a control siRNA. For Fig. 6C, cells were harvested for immunoblot analysis using an antibody to LXR that recognizes both LXRα and LXRβ protein. LXR protein abundance is significantly reduced by the LXR siRNA. Fig. 6D shows that treatment of adipocytes with LXR siRNA completely eliminated the increase in apoE mRNA levels in response to rosiglitazone treatment. Silencing LXR also completely prevented the PPARγ-mediated increase in apoE protein (Fig. 6E). We have previously shown that the TG accumulation that follows PPARγ agonist treatment of adipocytes is dependent on the expression of adipocyte apoE (6Huang Z.H. Reardon C.A. Mazzone T. Diabetes.. 2006; 55: 3394-3402Google Scholar). This latter observation, along with the results in Fig. 6E, supports the prediction that silencing LXR would prevent the PPARγ-mediated increase i
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