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• W2046604129 abstract "The α2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that interacts with a variety of molecules. It is implicated in biologically important processes such as lipoprotein metabolism, neurological function, tissue remodeling, protease complex clearance, and cell signal transduction. However, the regulation of LRP gene expression remains largely unknown. In this study, we have analyzed 2 kb of the 5′-flanking region of the LRP gene and identified a predicted peroxisome proliferator response element (PPRE) from −1185 to −1173. Peroxisome proliferator-activated receptor γ (PPARγ) ligands such as fatty acids and rosiglitazone increased functional cell surface LRP by 1.5–2.0-fold in primary human adipocytes and in the SW872 human liposarcoma cell line as assessed by activated α2-macroglobulin binding and degradation. These agents were found to increase LRP transcription. Gel shift analysis of the putative PPRE demonstrated direct binding of PPARγ/retinoid X receptor α heterodimers to the PPRE in the LRP gene. Furthermore, these heterodimers could no longer interact with a mutated PPRE probe. The isolated promoter was functional in SW872 cells, and its activity was increased by 1.5-fold with the addition of rosiglitazone. Furthermore, the isolated response element was similarly responsive to rosiglitazone when placed upstream of an ideal promoter. Mutagenesis of the predicted PPRE abolished the ability of this construct to respond to rosiglitazone. These data demonstrate that fatty acids and rosiglitazone directly stimulate transcription of the LRP gene through activation of PPARγ and increase functional LRP expression. The α2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that interacts with a variety of molecules. It is implicated in biologically important processes such as lipoprotein metabolism, neurological function, tissue remodeling, protease complex clearance, and cell signal transduction. However, the regulation of LRP gene expression remains largely unknown. In this study, we have analyzed 2 kb of the 5′-flanking region of the LRP gene and identified a predicted peroxisome proliferator response element (PPRE) from −1185 to −1173. Peroxisome proliferator-activated receptor γ (PPARγ) ligands such as fatty acids and rosiglitazone increased functional cell surface LRP by 1.5–2.0-fold in primary human adipocytes and in the SW872 human liposarcoma cell line as assessed by activated α2-macroglobulin binding and degradation. These agents were found to increase LRP transcription. Gel shift analysis of the putative PPRE demonstrated direct binding of PPARγ/retinoid X receptor α heterodimers to the PPRE in the LRP gene. Furthermore, these heterodimers could no longer interact with a mutated PPRE probe. The isolated promoter was functional in SW872 cells, and its activity was increased by 1.5-fold with the addition of rosiglitazone. Furthermore, the isolated response element was similarly responsive to rosiglitazone when placed upstream of an ideal promoter. Mutagenesis of the predicted PPRE abolished the ability of this construct to respond to rosiglitazone. These data demonstrate that fatty acids and rosiglitazone directly stimulate transcription of the LRP gene through activation of PPARγ and increase functional LRP expression. low density lipoprotein receptor-related protein peroxisome proliferator-activated receptor retinoid X receptor peroxisome proliferator response element fetal calf serum reverse transcription α2-macroglobulin activated α2M electrophoretic mobility shift assay(s) human fatty acyl CoA oxidase phosphate-buffered saline receptor-associated protein bovine serum albumin Hanks' balanced salt solution The α2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP)1 is a 600-kDa multifunctional endocytic receptor that belongs to the low density lipoprotein receptor gene family (1Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Google Scholar). LRP binds and internalizes a broad range of biologically diverse ligands. These include proteases of the fibrinolytic pathway (2Mikhailenko I. Kounnas M.Z. Strickland D.K. J. Biol. Chem. 1995; 270: 9543-9549Google Scholar) and serpin-enzyme complexes (3Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Google Scholar) as well as proteins important in lipoprotein metabolism such as lipoprotein lipase, hepatic lipase, lipoprotein(a), and apoE-rich lipoproteins (4Chappell D.A. Fry G.L. Waknitz M.A. Iverius P.H. Williams S.E. Strickland D.K. J. Biol. Chem. 1992; 267: 25764-25767Google Scholar, 5Kounnas M.Z. Chappell D.A. Wong H. Argraves W.S. Strickland D.K. J. Biol. Chem. 1995; 270: 9307-9312Google Scholar, 6Kuchenhoff A. Harrach-Ruprecht B. Robenek H. Am. J. Physiol. 1997; 272: C369-C382Google Scholar, 7Krapp A. Ahle S. Kersting S. Hua Y. Kneser K. Nielsen M. Gliemann J. Beisiegel U. J. Lipid Res. 1996; 37: 926-936Google Scholar, 8Medh J.D. Bowen S.L. Fry G.L. Ruben S. Andracki M. Inoue I. Lalouel J.M. Strickland D.K. Chappell D.A. J. Biol. Chem. 1996; 271: 17073-17080Google Scholar, 9Reblin T. Niemeier A. Meyer N. Willnow T.E. Kronenberg F. Dieplinger H. Greten H. Beisiegel U. J. Lipid Res. 1997; 38: 2103-2110Google Scholar). Targeted deletion of LRP in the mouse results in early embryonic death, demonstrating a critical function for LRP in prenatal development (10Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Google Scholar). LRP has also been shown to have a dual role in ॆ-amyloid metabolism by enhancing ॆ-amyloid precursor protein conversion to ॆ-amyloid (11Ulery P.G. Beers J. Mikhailenko I. Tanzi R.E. Rebeck G.W. Hyman B.T. Strickland D.K. J. Biol. Chem. 2000; 275: 7410-7415Google Scholar) and mediating the clearance of ॆ-amyloid (12Kang D.E. Pietrzik C.U. Baum L. Chevallier N. Merriam D.E. Kounnas M.Z. Wagner S.L. Troncoso J.C. Kawas C.H. Katzman R. Koo E.H. J. Clin. Invest. 2000; 106: 1159-1166Google Scholar, 13Shibata M. Yamada S. Kumar S.R. Calero M. Bading J. Frangione B. Holtzman D.M. Miller C.A. Strickland D.K. Ghiso J. Zlokovic B.V. J. Clin. Invest. 2000; 106: 1489-1499Google Scholar). These data support a potentially complex role for LRP in the pathogenesis of Alzheimer's disease (14Ulery P.G. Strickland D.K. J. Clin. Invest. 2000; 106: 1077-1079Google Scholar). In addition, LRP mediates signal transduction by interacting with cytosolic adaptor and scaffold proteins including DAB-1, JIP-2, and PSD-95 (15Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Google Scholar). A 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum-resident protein that functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity (16Battey F.D. Gafvels M.E. FitzGerald D.J. Argraves W.S. Chappell D.A. Strauss J.F. Strickland D.K. J. Biol. Chem. 1994; 269: 23268-23273Google Scholar, 17Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar, 18Willnow T.E. Armstrong S.A. Hammer R.E. Herz J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4537-4541Google Scholar). RAP is required for the proper folding and export of the LRP from the endoplasmic reticulum by preventing the premature binding of co-expressed ligands, such as apoE (19Obermoeller L.M. Warshawsky I. Wardell M.R. Bu G. J. Biol. Chem. 1997; 272: 10761-10768Google Scholar, 20Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. EMBO J. 1996; 15: 2632-2639Scopus (238) Google Scholar, 21Savonen R. Obermoeller L.M. Trausch-Azar J.S. Schwartz A.L. Bu G. J. Biol. Chem. 1999; 274: 25877-25882Google Scholar). RAP binds LRP directly via adjacent complement-type repeats, both containing a conserved acidic residue (22Andersen O.M. Christensen L.L. Christensen P.A. Sorensen E.S. Jacobsen C. Moestrup S.K. Etzerodt M. Thogersen H.C. J. Biol. Chem. 2000; 275: 21017-21024Google Scholar), and thus stearically interferes with binding of other LRP ligands including α2M* and remnant lipoproteins. LRP is expressed in a variety of cells with high expression in hepatocytes, macrophages, neuronal cells, fibroblasts, and adipocytes (23Herz J. Betterridge D.J. Illingworth D.R. Shepherd J. Lipoproteins in Health and Disease. Arnold, London1999: 333-359Google Scholar). In human adipocytes, LRP is involved in chylomicron remnant cholesterol clearance (24Descamps O. Bilheimer D. Herz J. J. Biol. Chem. 1993; 268: 974-981Google Scholar) and mediates the selective uptake of high density lipoprotein-derived cholesteryl ester (25Vassiliou G. Benoist F. Lau P. Kavaslar G.N. McPherson R. J. Biol. Chem. 2001; 276: 48823-48830Google Scholar). Despite the diverse and biologically important functions of LRP, relatively little is known about the regulation of LRP gene expression. The human LRP gene consists of 89 exons spanning 92 kb, encoding an mRNA of 15 kb (26Van Leuven F. Stas L. Hilliker C. Lorent K. Umans L. Serneels L. Overbergh L. Torrekens S. Moechars D. De Strooper B. Genomics. 1994; 24: 78-89Google Scholar). Although the coding regions of LRP and the low density lipoprotein receptor share some homology, there is little apparent similarity in their promoter regions. A portion of the 5′-flanking region of the LRP gene has been previously described (27Kutt H. Herz J. Stanley K.K. Biochim. Biophys. Acta. 1989; 1009: 229-236Google Scholar,28Gaeta B.A. Borthwick I. Stanley K.K. Biochim. Biophys. Acta. 1994; 1219: 307-313Google Scholar). In Chinese hamster ovary cells, the minimal promoter driving expression of the LRP gene was shown to be in a 1.6-kb GC-rich fragment that does not contain a classical TATA box. An Sp1 sequence at −80 and two clusters of Sp1 sequences between −520 and −752 were characterized and shown to be critical for expression of the gene. The promoter region also contains a consensus NRF-1 element located at −152 that may mediate the effects of cAMP and IFNγ (29Gafvels M.E. Coukos G. Sayegh R. Coutifaris C. Strickland D.K. Strauss J.F. J. Biol. Chem. 1992; 267: 21230-21234Google Scholar, 30Businaro R. Fabrizi C. Persichini T. Starace G. Ennas M.G. Fumagalli L. Lauro G.M. J. Neuroimmunol. 1997; 72: 75-81Google Scholar). There is a consensus sterol response element located at +233 in the 5′-untranslated region; however, studies have shown that LRP gene expression is not regulated by cholesterol (27Kutt H. Herz J. Stanley K.K. Biochim. Biophys. Acta. 1989; 1009: 229-236Google Scholar). We have studied the regulation of LRP gene expression during human preadipocyte differentiation and in response to free fatty acid availability. LRP mRNA was absent in human preadipocytes, and the appearance of LRP mRNA during differentiation coincided with that of the peroxisome proliferator-activated receptor γ (PPARγ). 2F. Benoist and R. McPherson, unpublished data. PPARγ is a transcription factor belonging to the nuclear hormone receptor superfamily. The retinoid X receptor α (RXRα) is the obligate partner of PPARγ (31Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Google Scholar), and together they form a heterodimer that regulates gene transcription following binding to a peroxisome proliferator response element (PPRE) and activation by specific ligands. The PPRE consists of a hexameric nucleotide repeat of the recognition motif (TGACCT) spaced by one nucleotide (DR-1) (32DiRenzo J. Soderstrom M. Kurokawa R. Ogliastro M.H. Ricote M. Ingrey S. Horlein A. Rosenfeld M.G. Glass C.K. Mol. Cell. Biol. 1997; 17: 2166-2176Google Scholar, 33Schoonjans K. Staels B. Auwerx J. J. Lipid Res. 1996; 37: 907-925Google Scholar). PPARγ is activated by a number of ligands including long chain fatty acids (34Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Google Scholar), prostaglandin J2 derivatives (35Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Google Scholar), and thiazolidenediones (36Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Google Scholar, 37De Vos P. Lefebvre A.M. Miller S.G. Guerre-Millo M. Wong K. Saladin R. Hamann L.G. Staels B. Briggs M.R. Auwerx J. J. Clin. Invest. 1996; 98: 1004-1009Google Scholar). The effects of PPARγ ligands on gene expression are direct results of increased transcription of the target gene containing a PPRE. In our own analysis of the LRP promoter, we have identified a novel sequence (TGAACTcTGACAT) in the 5′-flanking sequence at positions −1185 to −1173 with high homology to the PPRE. We report here that functional cell surface LRP is increased by PPARγ ligands via the activation of PPARγ transcriptional complexes that bind the newly identified PPRE in the LRP promoter. This is the first report demonstrating regulation of LRP gene expression via a discrete promoter element. Subcutaneous adipose tissue was collected from healthy normolipemic subjects undergoing reduction mammoplasty procedures. Preadipocytes were isolated and cultured from adipose tissue through collagenase digestion, centrifugation, and filtration as previously described (38Benoist F. Lau P. McDonnell M. Doelle H. Milne R. McPherson R. J. Biol. Chem. 1997; 272: 23572-23577Google Scholar, 39Radeau T. Robb M. Lau P. Borthwick J. McPherson R. Atherosclerosis. 1998; 139: 369-376Google Scholar, 40Radeau T. Robb M. McDonnell M. McPherson R. Biochim. Biophys. Acta. 1998; 1392: 245-253Google Scholar, 41Radeau T. Lau P. Robb M. McDonnell M. Ailhaud G. McPherson R. J. Lipid Res. 1995; 36: 2552-2561Google Scholar). The preadipocytes were cultured in differentiation media for 10–14 days. The cells were then insulin-starved in the presence or absence of varying concentrations of the PPARγ ligand rosiglitazone for 24 h prior to assays. The control cells were treated with vehicle only (Me2SO). The human liposarcoma cell line SW872 (American Type Culture Collection, Manassas, VA) was previously characterized and has been shown to be a good cell model for adipocyte gene expression (42Gauthier B. Robb M. Gaudet F. Ginsburg G.S. McPherson R. J. Lipid Res. 1999; 40: 1284-1293Google Scholar, 43Richardson M.A. Berg D.T. Johnston P.A. McClure D. Grinnell B.W. J. Lipid Res. 1996; 37: 1162-1166Google Scholar, 44Izem L. Morton R.E. J. Biol. Chem. 2001; 276: 26534-26541Google Scholar). The cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (3:1) (Invitrogen) supplemented with 57 fetal bovine serum and 17l-glutamine (Invitrogen), 10 mmNaCO3, and 50 ॖg/ml gentamycin (NovoPharm, Toronto, Canada) in the presence of 57 CO2 at 37 °C. Lipoprotein-deficient fetal calf serum was prepared as described previously (45Schumaker V.N. Puppione D.L. Methods Enzymol. 1986; 128: 155-170Google Scholar) and dialyzed against PBS for 24 h. The effect of fatty acids and thiazolidenediones on LRP mRNA and protein levels was determined by incubating cells for 24 h in medium containing either lipoprotein-deficient fetal calf serum or CS in the presence or absence of oleic acid (18:1) or arachidonic acid (20:4) (Sigma) or rosiglitazone (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA). All of the conditions were studied in triplicate. SW872 cells were cultured for 24 h with either 160 ॖmarachidonic acid or 500 nm rosiglitazone in media containing CS prior to measuring their ability to degrade125I-labeled α2M* as previously described (46Vassiliou G. Stanley K.K. J. Biol. Chem. 1994; 269: 15172-15178Google Scholar). Differentiated primary human adipocytes cultured in differentiating medium were starved of insulin in the presence or absence of increasing concentrations of rosiglitazone prior to measuring their ability to degrade or bind 125I-α2M* (46Vassiliou G. Stanley K.K. J. Biol. Chem. 1994; 269: 15172-15178Google Scholar). Control cells were treated with vehicle only (Me2SO). As a further control, the cells were treated in the presence or absence of RAP because this molecule will impair LRP function. To determine the transcriptional effect of rosiglitazone, 500 nm of this ligand was added to cells cultured as described above in the presence or absence of 10 ॖg/ml α-amanitin (Sigma), a potent inhibitor of RNA polymerase II (47Adolph S. Brusselbach S. Muller R. J. Cell Sci. 1993; 105: 113-122Google Scholar). Total cellular RNA was isolated from both differentiated primary human adipocytes and SW872 cells with Tri-Reagent (Bio/Can, Mississauga, Canada) according to the manufacturer's instructions. RNA samples from differentiated primary adipocytes that were to be used in RT-PCR reactions were treated with amplification grade DNase I to deplete the samples of any DNA contamination according to the manufacturer's instructions (Invitrogen). RNA concentration was determined spectrophotometrically usingA260/280. Total RNA (5 ॖg) was separated by agarose gel electrophoresis using the NorthernMax-Gly kit and transferred to BrightStar-Plus nylon membrane according to the manufacturer's instructions (Ambion, Austin, TX). DNA probes were synthesized by RT-PCR; first strand DNA was synthesized as described below, and PCR was performed using the following primers: LRPf, 5′-GAGTACCAGGTCCTGTACATCGCTG-3′, and LRPr, 5′-CTCGTCAATCATGCCCGAGATGAGC-3′; ॆ-actin-f, 5′-GCCCCTCCATCGTCCACCGC-3′, and ॆ-actin-r, 5′-GGGCACGAAGGCTCATCATT-3′. The PCR products were gel-purified using the QiaexII kit (Qiagen), and the purified DNA was subsequently labeled using the Rediprime II random prime labeling kit according the manufacturer's instructions (Amersham Biosciences). The probes were cleaned up with NICK columns (Amersham Biosciences), and the specific activity was determined by use of a scintillation counter. Hybridizations and washes were performed according the NorthernMax-Gly kit instructions (Ambion). RT-PCR was performed using a two-step approach. First strand cDNA was synthesized using 2.5 ॖg of total RNA, 10 ॖm random decamer primers (Ambion), and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and incubated at 42 °C for 1 h. Consecutive PCR reactions were then performed on the first strand cDNA using the primers shown below and the SYBR green 舠taq-start舡 polymerase and the LightCycler Apparatus according the manufacturer's instructions (Roche Molecular Biochemicals). The data from the LightCycler was repeated using relative quantitative RT-PCR as described below. For SW 872 samples, relative quantitative RT-PCR was performed using the Quantum RNA 18 S Internal Standards kit from Ambion. This kit has been previously shown to allow the accurate determination of relative changes in gene expression between samples (48Dodd F. Limoges M. Boudreau R.T. Rowden G. Murphy P.R. Too C.K. J. Cell. Biochem. 2000; 77: 624-634Google Scholar). Briefly, first strand cDNA was synthesized using 2.5 ॖg of total RNA, 10 ॖm random decamer primers (Ambion), and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and incubated at 42 °C for 1 h. LRP forward primer (5′-GAGTACCAGGTCCTGTACATCGCTG-3′) and reverse primer (5′-CTCGTCAATCATGCCCGAGATGAGC-3′) were designed to amplify a region of the LRP mRNA that is ∼400 bp in size, whereas the primers provided in the 18 S Internal Standards kit produced a band that is ∼500 bp. A cycle number of 23 was determined to be within the linear range of PCR and was used for all subsequent PCR reactions. The 18 S primer:competimer ratio of 3:7 was experimentally determined so that the LRP and 18 S PCR products were amplified to give similar yields so that they could be compared between samples. PCR was performed on 1 ॖl of the RT reaction using 20 pmol of each LRP primer and 4 ॖl of 18 S primer/competitor mix with the following PCR conditions; 1 cycle of 95 °C for 3 min and 23 cycles of 95 °C for 30 s, 66 °C for 30 s, and 72 °C for 30 s. Cocktails containing all shared components were used to reduce variation between samples. The PCR products were subjected to electrophoresis through a 1.57 agarose gel and visualized with ethidium bromide staining. The band intensities were measured using the ChemiDoc apparatus and Quantity One software (Bio-Rad). Relative intensity was calculated by dividing the 400-bp band corresponding to the LRP message by the 488-bp band corresponding to the 18 S message. The cells were cultured on 35-mm cover glass bottom dishes (MatTek, Ashland, MA) as described above and supplemented with 160 ॖm arachidonic acid, 500 nm rosiglitazone, or control. α2M was activated by incubating purified α2M with 400 mm methylamine for 16 h at room temperature. α2M* was fluorescently labeled using Cy3 monofunctional reactive dye (Amersham Biosciences) to a dye:protein ratio of 1.3 according to the manufacturer's instructions. The cells were placed on ice for 1 h in 3:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium supplement with 2 mg/ml BSA buffered with 10 mm HEPES. Labeled α2M* was diluted in the same medium to a concentration of 1 ॖg/ml and was added to the cells at 0 °C for 45 min. The cells were washed with ice-cold PBS three times prior to being fixed with 47 paraformaldehyde for 10 min at 0 °C. The cells were rinsed with PBS and kept in 2 ml of PBS at room temperature for fluorescence microscopy. Binding was competed with 30-fold excess of unlabelled α2M*. The cells were viewed with an Olympus IX50 fluorescent microscope, and the images were taken using a coded CCD camera (MicroMax) and WinView software from Princeton Instruments (Princeton, NJ). Total cellular protein (5 ॖg) from SW872 cells incubated in the presence or absence of various PPARγ ligands was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose (49Hames B.D. Hames B.D. Rickwood D. Gel Electrophoresis of Proteins: A Practical Approach. Oxford University Press, Oxford, UK1981: 1-86Google Scholar). The LRP was detected (49Hames B.D. Hames B.D. Rickwood D. Gel Electrophoresis of Proteins: A Practical Approach. Oxford University Press, Oxford, UK1981: 1-86Google Scholar) using a polyclonal rabbit antisera (from Dr. G. Bu) followed by chemiluminescent detection (Pierce) of a secondary antibody conjugated to horseradish peroxidase. The blot was developed, and the bands were quantified using the ChemiDoc apparatus and Quantity One software (Bio-Rad). Triplicate cell samples were processed and are summarized in the graph. Molecular biology techniques were essentially as described by Sambrook et al. (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The nuclear proteins were extracted from SW872 cells as previously described (51Slomiany B.A. Kelly M.M. Kurtz D.T. BioTechniques. 2000; 28: 938-942Google Scholar) or from primary human adipocytes as described (52Dugail I. Ailhaud G. Adipose Tissue Protocols. Humana Press, Totawa, NJ2001: 141-147Google Scholar). Protein concentration was determined using BCA protein reagent (BioLynx, Brockville, Canada) according to the manufacturer's instructions. Double-stranded oligonucleotides (oligomers) corresponding to the PPRE of LRP (5′-CCCCGCTCCTTGAACTCTGACATCGAGACACCTA-3′) were radioactively end-labeled with [γ-32P]dATP (Amersham Biosciences) using T4 polynucleotide kinase (Invitrogen) and purified from unincorporated nucleotides by gel filtration through G-50 spin columns (Amersham Biosciences). The same procedure was used for oligomers corresponding to the PPRE of the human fatty acyl CoA oxidase gene (hACOX) (5′-TCCGAACGTGACCTTTGTCCTGGTCCCCTTT-3′) and oligomers corresponding to the mutated form of the LRP PPRE (the mutated half-site is underlined) (5′-CCCCGCTCCTTGAACTCAACGATCGAGACACC TA-3′). The specific activities of the oligomers were ∼250 cpm/fmol. These were diluted to 60 fmol/ॖl for use in the assay. PPARγ (from Dr. Bruce Spiegelman) was subcloned into the MluI andNotI sites of pSPORT1 (Invitrogen) using PCR-based methods. RXRα was provided by Dr. Michael Saunders in pSG5. Both constructs are driven by the T7 RNA polymerase promoter for use in the TNT T7-coupled reticulocyte lysate system (Promega) for in vitrotranscription/translation. All of the EMSA reactions were carried out on ice in 20 ॖl of binding buffer (12.5 mm HEPES-KOH, pH 7.6, 6 mm MgCl2, 5.5 mm EDTA, and 50 mm KCl) supplemented with 5 mmdithiothreitol, 0.25 ॖg of low fat milk, 0.05 ॖg of poly(dI-dC), and 107 glycerol. For EMSA reactions with TNT-purified proteins, 2 ॖl of the TNT reaction was added to the reaction mix along with 1 ॖl (60 fmol) of labeled oligomers. For EMSA reactions with nuclear protein extracts, 6 ॖl of nuclear extracts were added to the reaction mix with 1 ॖl (60 fmol) of labeled oligomers. These reactions were left on ice for 20 min. Following the 20-min incubation, 2 ॖl of 207 Ficoll was added to the samples. DNA-protein complexes were then resolved by electrophoresis through 67 polyacrylamide gels in 0.25× Tris borate running buffer (20 mm Tris borate, pH 7.2, 0.5 mm EDTA). Supershift assays were performed using the PPARγ NuShift kit following the manufacturer's protocol (Active Motif). The nonspecific antibody used was mouse monoclonal anti-ॆ-actin (Santa-Cruz). Complementary primers with flanking NheI and XhoI restriction sites (5′-CTAGCCTCCTTGAACTCTGACATGCAGACC-3′) were annealed and subcloned into the luciferase reporter vector, pGL3-Promoter (Promega). This new vector contains a single copy of the putative PPRE upstream of an ideal promoter and is designated pGL3-PPRE. We also prepared 1.9 kb of the 5′-flanking region of LRP by PCR amplification from the LRP-BAC construct prepared by Dr. Jan Boren (53Boren J. Lee I. Callow M.J. Rubin E.M. Innerarity T.L. Genome Res. 1996; 6: 1123-1130Google Scholar), which contains the entire 92-kb gene of human LRP, using the primers 5′-GCAACGAGCTCCGTAAAAGGGGGAAG-3′ and 5′-GCAGCAGATCTTTCCCCGGACTGAAG-3′. This fragment was subcloned into theSacI and BglII sites of the luciferase reporter vector, pGL3-Basic (Promega), and designated pGL3-LRP. Mutagenesis of the PPRE was performed by PCR using PFUTurbo (Stratagene, La Jolla, CA) according to their Quikchange site-directed mutagenesis protocol. The complementary primers (5′-CCCGCTCCTTGAACTCAACGATGCAGACACC-3′) were designed to mutate a single half-site of the PPRE so that PPARγ would no longer bind the response element (mutated nucleotides are underlined). This construct was designated pGL3- LRPmutant PPRE. Both pGL3-LRP and pGL3-LRPmutant PPRE were sequenced to confirm that the promoter sequence was correct (compared with GenBankTM accession number Y18524) and to verify the mutagenesis. Confluent SW872 cells were trypsinized and seeded at a density of 1.25 × 105cells/well in 12-well plates 48 h prior to transfection. The cells were ∼70–807 confluent at the time of transfection. Fresh medium containing CS was added 12 h preceding transfections. The cells were co-transfected with 4 ॖg of the firefly luciferase reporter vector (either pGL3-basic, pGL3-LRP, PGL3-PPRE, or pGL3-LRPmutant PPRE) and 0.25 ॖg of theRenilla luciferase-bearing reporter vector, pRL-CMV (Promega) using the calcium phosphate-DNA precipitate method (54Blackhart B.D. Yao Z.M. McCarthy B.J. J. Biol. Chem. 1990; 265: 8358-8360Google Scholar). The cells were shocked with 157 glycerol for 2 min, 4 h after the transfection, and washed three times with PBS before the addition of medium. The cells were treated 12 h later with Me2SO alone (vehicle control) or varying concentrations of rosiglitazone. After 24 h (36 h total post-transfection) the cells were scraped in 250 ॖl of reporter lysis buffer (Promega) and kept on ice until assayed. Luciferase activities derived from both firefly (LRP constructs) andRenilla (pRL-CMV) proteins were measured using the dual luciferase reporter assay system (Promega) and recorded using a Monolight 2010c luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). Renilla luciferase activity was then used to standardize for transfection efficiency. The results are expressed as the means ± S.E. Where indicated, the statistical significance of the differences between groups was determined using Student's ttest or analysis of variance. The effects of PPARγ ligands on differentiated primary human adipocytes were examined by an 125I-α2M* binding assay (Fig. 1A). TheBmax of cells incubated with 1 ॖmrosiglitazone (27.0) is ∼1.5 times greater than that of the vehicle-treated cells (17.3), indicating that there is an increase in the levels of functional cell surface LRP. The difference in binding was found to be highly significant with a two-tailed p value of less than 0.0001. When the cells were treated with RAP (30 ॖg/ml), the amount of binding was reduced to background levels, demonstrating that this process is LRP-specific. There was no statistically significant difference in the Kd between the treated and control cells as illustrated in the Scatchard plot (Fig.1B), indicating that the binding affinities have not changed. 125I-α2M* degradation assays were also performed in the presence or absence of PPARγ ligands for the differentiated primary human adipocytes and SW872 cells. In primary adipocytes, there was a direct relationship between the amount of rosiglitazone added and the amount of α2M* degradation over 8 h (Fig.2A) with very significant increases ranging from 1.2- to 1.7-fold over control (p< 0.009). The RAP is an antagonist of all identified LRP ligands including α2M*; therefore we used purified RAP to block LRP function in our assays. When cells were treated with RAP, the amou" @default.
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• W2046604129 date "2003-04-01" @default.
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• W2046604129 title "Adipocyte Low Density Lipoprotein Receptor-related Protein Gene Expression and Function Is Regulated by Peroxisome Proliferator-activated Receptor γ" @default.
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• W2046604129 doi "https://doi.org/10.1074/jbc.m212989200" @default.
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