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• W2165429105 abstract "Certain triglyceride-rich lipoproteins (TRLs), specifically chylomicrons, dyslipemic VLDLs, and their remnants, are atherogenic and can induce monocyte-macrophage foam cell formation in vitro via the apolipoprotein B-48 receptor (apoB-48R). Human atherosclerotic lesion foam cells express the apoB-48R, as determined immunohistochemically, suggesting it can play a role in the conversion of macrophages into foam cells in vivo. The regulation of the apoB-48R in monocyte-macrophages is not fully understood, albeit previous studies indicated that cellular sterol levels and state of differentiation do not affect apoB-48R expression. Since peroxisome proliferator-activated receptors (PPARs) regulate some aspects of cellular lipid metabolism and may be protective in atherogenesis by up-regulation of liver X-activated receptor α and ATP-binding cassette transporter A1, we examined the regulation of apoB-48R by PPAR ligands in human monocyte-macrophages. Using real-time PCR, Northern, Western, and functional cellular lipid accumulation assays, we show that PPARα and PPARγ activators significantly suppress the expression of apoB-48R mRNA in human THP-1 and blood-borne monocyte-macrophages. Moreover, PPAR activators inhibit the expression of the apoB-48R protein and, notably, the apoB-48R-mediated lipid accumulation of TRL by THP-1 monocytes in vitro.If PPAR activators also suppress the apoB-48R pathway in vivo, diminished apoB-48R-mediated monocyte-macrophage lipid accumulation may be yet another antiatherogenic effect of the action of PPAR ligands. Certain triglyceride-rich lipoproteins (TRLs), specifically chylomicrons, dyslipemic VLDLs, and their remnants, are atherogenic and can induce monocyte-macrophage foam cell formation in vitro via the apolipoprotein B-48 receptor (apoB-48R). Human atherosclerotic lesion foam cells express the apoB-48R, as determined immunohistochemically, suggesting it can play a role in the conversion of macrophages into foam cells in vivo. The regulation of the apoB-48R in monocyte-macrophages is not fully understood, albeit previous studies indicated that cellular sterol levels and state of differentiation do not affect apoB-48R expression. Since peroxisome proliferator-activated receptors (PPARs) regulate some aspects of cellular lipid metabolism and may be protective in atherogenesis by up-regulation of liver X-activated receptor α and ATP-binding cassette transporter A1, we examined the regulation of apoB-48R by PPAR ligands in human monocyte-macrophages. Using real-time PCR, Northern, Western, and functional cellular lipid accumulation assays, we show that PPARα and PPARγ activators significantly suppress the expression of apoB-48R mRNA in human THP-1 and blood-borne monocyte-macrophages. Moreover, PPAR activators inhibit the expression of the apoB-48R protein and, notably, the apoB-48R-mediated lipid accumulation of TRL by THP-1 monocytes in vitro. If PPAR activators also suppress the apoB-48R pathway in vivo, diminished apoB-48R-mediated monocyte-macrophage lipid accumulation may be yet another antiatherogenic effect of the action of PPAR ligands. Elevated levels of plasma triglycerides (TGs) and VLDL, persistent dietary chylomicrons (CMs), and their remnants are risk factors for cardiovascular disease, a major cause of death worldwide (1Hokanson J.E. Austin M.A. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies.J. Cardiovasc. Risk. 1996; 3: 213-219Google Scholar). Atherogenesis involves both endothelial cell dysfunction and the appearance of lipid-filled foam cells, primarily of macrophage origin, in the arterial intima throughout the atherosclerotic process. The TG-rich lipoproteins (TRLs), hypertriglyceridemic (HTG) VLDL, CMs, and their remnants are the only native, nonmodified lipoproteins that cause rapid receptor-mediated macrophage lipid engorgement in vitro; normal VLDL and LDL do not (2Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis.Annu. Rev. Biochem. 1983; 52: 223-261Google Scholar, 3Gianturco S.H. Bradley W.A. Gotto A.M. Morrisett J.D. Peavy D.L. Hypertriglyceridemic very low density lipoproteins induce triglyceride synthesis and accumulation in mouse peritoneal macrophages.J. Clin. Invest. 1982; 70: 168-178Google Scholar). Indeed, THP-1 monocyte-macrophages and native blood-borne macrophages show massive lipid accumulation (3- to 10-fold increase in TG mass) in ∼4 h when exposed to physiological levels of these TRLs in vitro via the apolipoprotein B-48 receptor (apoB-48R) described below (4Brown M.L. Ramprasad M.P. Umeda P.K. Tanaka A. Kobayashi Y. Watanabe T. Shimoyamada H. Kuo W.L. Li R. Song R. Bradley W.A. Gianturco S.H. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 7488-7493Google Scholar). Similar lipid accumulation occurs in vivo: humans with persistently elevated CMs have foam cells in their bone marrow, spleen, liver, and skin (5Fredrickson D.S. Goldstein J.L. Brown M.S. The familial hyperlipoproteinemias.in: Stanbury J.G. Wyngaarden M.F. Fredrickson D.S. The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York1978: 604-655Google Scholar). As these TRLs also cause endothelial cell cholesterol uptake (6Gianturco S.H. Eskin S.G. Navarro L.T. Lahart C.J. Smith L.C. Gotto A.M. Abnormal effects of hypertriacylglycerolemic very low-density lipoproteins on 3-hydroxy-3-methylglutaryl-CoA reductase activity and viability of cultured bovine aortic endothelial cells.Biochim. Biophys. Acta. 1980; 618: 143-152Google Scholar, 7Fielding C.J. Vlodavsky I. Fielding P.E. Gospodarowicz D. Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.J. Biol. Chem. 1979; 254: 8861-8868Google Scholar) and fibrinolytic dysfunction in vitro (8Li X-N. Kroon J.C. Benza R.L. Parks J.M. Varma V.K. Bradley W.A. Gianturco S.H. Taylor K.B. Grammer J.R. Tabengwa E.M. Booyse F.M. Hypertriglyceridemic VLDL decreases plasminogen binding to endothelial cells and surface-localized fibrinolysis.Biochemistry. 1996; 35: 6080-6088Google Scholar), TRLs may be atherothrombogenic in subjects with elevated fasting and postprandial plasma TG.TRLs interact with cells by many described mechanisms. HTG-VLDL and CM remnants bind to the LDL receptor and gene family members via apoE (9Gianturco S.H. Gotto A.M. Hwang S.L. Karlin J.B. Lin A.H. Prasad S.C. Bradley W.A. Apolipoprotein E mediates uptake of Sf 100–400 hypertriglyceridemic very low density lipoproteins by the low density lipoprotein receptor pathway in normal human fibroblasts.J. Biol. Chem. 1983; 258: 4526-4533Google Scholar, 10Bradley W.A. Hwang S.L. Karlin J.B. Lin A.H. Prasad S.C. Gotto A.M. Gianturco S.H. Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins.J. Biol. Chem. 1984; 259: 14728-14735Google Scholar, 11Beisiegel U. Weber W. Ihrke G. Herz J. Stanley K.K. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein.Nature. 1989; 341: 162-164Google Scholar, 12Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins.Proc. Natl. Acad. Sci. USA. 1989; 86: 5810-5814Google Scholar). Diet-derived TRLs lack the C-terminal domain of the apoB-100 that binds to the LDL receptor (13Yang C.Y. Chen S.H. Gianturco S.H. Bradley W.A. Sparrow J.T. Tanimura M. Li W.H. Sparrow D.A. DeLoof H. Rosseneu M. Lee F.S. Gu Z.W. Gotto A.M. Chan L. Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100.Nature. 1986; 323: 738-742Google Scholar) and therefore cannot bind to the LDL receptor via apoB-48, the major apoB species formed in the intestine. Although the majority of CM is lipolyzed into remnants that are cleared by the liver via apoE (14Havel R.J. George Lyman Duff memorial lecture. Role of the liver in atherosclerosis.Arteriosclerosis. 1985; 5: 569-580Google Scholar, 15Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Google Scholar), a small but significant fraction of CM seemed to be cleared directly by reticuloendothelial cells, such as accessible macrophages in bone marrow and spleen, independent of apoE (16Nagata Y. Zilversmit D.B. Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate.J. Lipid Res. 1987; 28: 684-692Google Scholar, 17Hussain M.M. Mahley R.W. Boyles J.K. Fainaru M. Brecht W.J. Lindquist P.A. Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits: factors that modify tissue-specific uptake.J. Biol. Chem. 1989; 264: 9571-9582Google Scholar, 18Hussain M.M. Mahley R.W. Boyles J.K. Lindquist P.A. Brecht J.W. Innerarity T.L. Chylomicron metabolism. Chylomicron uptake by bone marrow in different animal species.J. Biol. Chem. 1989; 264: 17931-17938Google Scholar).The apoB-48R is an apoE-independent receptor in human and murine monocyte-macrophages (19Gianturco S.H. Lin A.H. Hwang S.L. Young J. Brown S.A. Via D.P. Bradley W.A. Distinct murine macrophage receptor pathway for human triglyceride-rich lipoproteins.J. Clin. Invest. 1988; 82: 1633-1643Google Scholar, 20Gianturco S.H. Ramprasad M.P. Lin A.H. Song R. Bradley W.A. Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.J. Lipid Res. 1994; 35: 1674-1687Google Scholar). Transfection of the apoB-48R minigene into Chinese hamster ovary cells in vitro confers all the known properties of the apoB-48R characterized in human monocytes and macrophages, including converting these cells in vitro into a foam cell phenotype upon challenge with TRL, with identical kinetic and saturation characteristics as seen in macrophages (4Brown M.L. Ramprasad M.P. Umeda P.K. Tanaka A. Kobayashi Y. Watanabe T. Shimoyamada H. Kuo W.L. Li R. Song R. Bradley W.A. Gianturco S.H. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 7488-7493Google Scholar). The apoB-48R binds apoB-48 of dietary TRL to a like domain of apoB-100 in HTG-VLDL (21Gianturco S.H. Ramprasad M.P. Song R. Li R. Brown M.L. Bradley W.A. Apolipoprotein B-48 or its apolipoprotein B-100 equivalent mediates the binding of triglyceride-rich lipoproteins to their unique human monocyte-macrophage receptor.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 968-976Google Scholar) and may account, in part, for the observed direct reticuloendothelial uptake of CM in vivo (16Nagata Y. Zilversmit D.B. Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate.J. Lipid Res. 1987; 28: 684-692Google Scholar, 17Hussain M.M. Mahley R.W. Boyles J.K. Fainaru M. Brecht W.J. Lindquist P.A. Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits: factors that modify tissue-specific uptake.J. Biol. Chem. 1989; 264: 9571-9582Google Scholar, 18Hussain M.M. Mahley R.W. Boyles J.K. Lindquist P.A. Brecht J.W. Innerarity T.L. Chylomicron metabolism. Chylomicron uptake by bone marrow in different animal species.J. Biol. Chem. 1989; 264: 17931-17938Google Scholar), and for foam cell formation seen in humans with elevated TRL (5Fredrickson D.S. Goldstein J.L. Brown M.S. The familial hyperlipoproteinemias.in: Stanbury J.G. Wyngaarden M.F. Fredrickson D.S. The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York1978: 604-655Google Scholar). Moreover, the apoB-48R likely participates in the foam cell formation in atherosclerosis-susceptible apoE-null mice, which have elevated apoB-48-containing lipoproteins (22Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Google Scholar, 23Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar, 24Brown M.L. Yui K. Smith J.D. LeBoeuf R.C. Weng W. Umeda P.K. Li R. Song R. Gianturco S.H. Bradley W.A. The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor.J. Lipid Res. 2002; 43: 1181-1191Google Scholar).Peroxisome proliferator activated receptors (PPARs) have been implicated in macrophage biology, lipid homeostasis, and atherogenesis. PPARs are ligand-activated transcription factors which, upon heterodimerization with the 9-cis-retinoic acid receptor, bind to specific peroxisome proliferator response elements (PPREs), thus regulating the expression of target genes involved in intra- and extracellular lipid metabolism. The naturally occurring prostaglandin 15-deoxy Δ12, 14-prostaglandin J2 (15-d-PGJ2), and the synthetic antidiabetic glitazones are ligands for PPARγ, while hypolipidemic fibrates are synthetic ligands for PPARα (25Barbier O. Torra I.P. Duguay Y. Blanquart C. Fruchart J.C. Glineur C. Staels B. Pleiotropic actions of peroxisome proliferator-activated receptors in lipid metabolism and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 717-726Google Scholar). PPARα is expressed in human monocytes and in fully differentiated macrophages (26Wahli W. Braissant O. Desvergne B. Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more.Chem. Biol. 1995; 2: 261-266Google Scholar), while PPARγ is expressed in cells undergoing differentiation into macrophages (27Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation.Nature. 1998; 391: 79-82Google Scholar, 28Li A.C. Brown K.K. Silvestre M.J. Willson T.M. Palinski W. Glass C.K. Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice.J. Clin. Invest. 2000; 106: 523-531Google Scholar, 29Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL.Cell. 1998; 93: 241-252Google Scholar, 30Chinetti G. Lestavel S. Bocher V. Remaley A.T. Neve B. Torra I.P. Teissier E. Minnich A. Jaye M. Duverger N. Brewer H.B. Fruchart J.C. Clavey V. Staels B. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.Nat. Med. 2001; 7: 53-58Google Scholar). In addition, both PPARα and PPARγ are detected in macrophage-rich regions of human atherosclerotic lesions (27Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation.Nature. 1998; 391: 79-82Google Scholar, 31Marx N. Sukhova G. Murphy C. Libby P. Plutzky J. Macrophages in human atheroma contain PPARgamma: differentiation-dependent peroxisomal proliferator-activated receptor gamma(PPARgamma) expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro.Am. J. Pathol. 1998; 153: 17-23Google Scholar, 32Chinetti G. Gbaguidi F.G. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. Staels B. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. [In Process Citation].Circulation. 2000; 101: 2411-2417Google Scholar). In macrophages, PPARs inhibit inflammatory cytokine-induced activation (33Jiang C. Ting A.T. Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines.Nature. 1998; 391: 82-86Google Scholar), promote apoptosis, and control lipid homeostasis through their effects on the expression of several key genes, including scavenger family members class A macrophage receptor (SR-A), class B scavenger receptor CD36, scavenger receptor class B type I, and cholesterol transporter ATP binding cassette transporter A1 (ABCA1) (28Li A.C. Brown K.K. Silvestre M.J. Willson T.M. Palinski W. Glass C.K. Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice.J. Clin. Invest. 2000; 106: 523-531Google Scholar, 29Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL.Cell. 1998; 93: 241-252Google Scholar, 30Chinetti G. Lestavel S. Bocher V. Remaley A.T. Neve B. Torra I.P. Teissier E. Minnich A. Jaye M. Duverger N. Brewer H.B. Fruchart J.C. Clavey V. Staels B. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.Nat. Med. 2001; 7: 53-58Google Scholar, 32Chinetti G. Gbaguidi F.G. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. Staels B. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. [In Process Citation].Circulation. 2000; 101: 2411-2417Google Scholar). The relative contributions of these PPAR-regulated genes in atherosclerotic processes remain unclear, although on balance PPAR ligands appear to inhibit atherogenesis in animal models.Since the apoB-48 receptor is involved in lipid metabolism in monocyte-macrophages, we hypothesized that PPAR might regulate its expression in macrophages. Here we report that PPAR ligands, somewhat surprisingly, suppress, rather than activate, expression of the macrophage apoB-48R mRNA and protein. Furthermore, our results indicate that the down-regulation of apoB-48R expression by PPAR activators in human monocyte-macrophages is accompanied by a parallel diminished cellular uptake of TRL, independent of apoE. If similar phenomena occur in vivo, the suppression of the apoB-48R pathway may contribute to the beneficial effects of PPAR agonists on atherogenesis by diminishing macrophage lipid accumulation via this pathway, particularly in hypertriglyceridemia.METHODSReagentsPioglitazone was kindly provided by Takeda Pharmaceutical Co., Osaka, Japan, and troglitazone was from Sankyo Pharmaceutical Co., Osaka, Japan. Wy14643 was purchased from Sigma (St. Louis, MO). 15-d-PGJ2 was from Cayman Chemical (Ann Arbor, MI).CellsTHP-1 cells were maintained in RPMI-1640 containing 10% FBS, l-glutamine, penicillin (100 U/ml), and streptomycin (10 μg/ml). THP-1 monocytes were differentiated to macrophages by the addition of phorbol ester as previously described (20Gianturco S.H. Ramprasad M.P. Lin A.H. Song R. Bradley W.A. Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.J. Lipid Res. 1994; 35: 1674-1687Google Scholar). Human peripheral blood-borne monocytes were isolated using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) (34Peper R.J. Tina W.Z. Mickelson M.M. Purification of lymphocytes and platelets by gradient centrifugation.J. Lab. Clin. Med. 1968; 72: 842-848Google Scholar) and then followed with the DYNAL monocyte-negative isolation kit (DYNAL A.S., Oslo, Norway) using the enriched monocyte fraction per the manufacturer's protocol.Northern blot analysesTotal RNA (20 μg per sample) was separated by agarose gel electrophoresis, transferred to nylon membranes (4Brown M.L. Ramprasad M.P. Umeda P.K. Tanaka A. Kobayashi Y. Watanabe T. Shimoyamada H. Kuo W.L. Li R. Song R. Bradley W.A. Gianturco S.H. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 7488-7493Google Scholar), and detected with human apoB-48R-, CD36-, and GAPDH-radiolabeled probes generated using RediPrime (Amersham Bioscience, Uppsala, Sweden) according to the manufacturer's instructions.RNA isolation and real-time quantitative RT-PCRTotal RNA was isolated from the monocytes using RNAzol B (Tel-Test, Friendswood, TX). Genomic DNA was removed by treatment with RNase-free DNase. The method of RT-PCR is briefly outlined here. One microgram of total RNA was reverse-transcribed in 1× PCR buffer using 10 U/μl reverse transcriptase (Life Technologies, Grand Island, NY) in the presence of 5 mM MgCl2, 1 mM dNTPs, 1 U/μl RNase inhibitor, and 2.5 mM random hexanucleotide primers. The RT reaction was carried out at 42°C for 50 min, followed by denaturation at 99°C for 5 min and cooling at 4°C for 5 min. The primers used in these studies are illustrated in Table 1. Quantitative real-time PCR was performed in a 20 μl reaction mixture (PCR kit from Roche Diagnostics) that contained 3 μl of reverse-transcribed cDNA, 5 μM forward primers, and 5 μM reverse primers. For each sample, triplicate analyses were performed. The resulting relative increase in reporter fluorescent dye emission, SYBR Green I, was monitored by the LightCycler system (Roche) using a protocol to reduce primer-dimer background interference (35Chou Q. Russell M. Birch D.E. Raymond J. Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.Nucleic Acids Res. 1992; 20: 1717-1723Google Scholar). The level of the apoB-48R and ABCA1 mRNA, relative to human β-actin, was calculated. The data are expressed as the ratio of target mRNA (relative to internal control) obtained from cells pretreated with PPAR ligands relative to mRNA obtained from cells treated with vehicle only.TABLE 1Primers for RT-PCRHuman apoB-48RSenseGGC-CTT-AGA-GGC-AGC-CAA-AAAntisenseTTC-CCA-GCT-TCT-CAG-CCT-CTHuman β-actinSenseTCA-CCC-ACA-CTG-TGC-CCA-TCT-ACG-AAntisenseCAG-CGG-AAC-CGC-TCA-TTG-CCA-ATG-GHuman ATP-binding cassette transporter A1SenseTGT-CCA-GTC-CAG-TAA-TGG-TTC-TGT-GTAntisenseGCG-AGA-TAT-GGT-CCG-GAT-TG Open table in a new tab Western blot analysis of human apoB-48RMonocytes (∼106 cells) were disrupted in the lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mM EDTA, 1% Triton X-114, and incubated with rotation for 30 min. After centrifugation of the sample to remove insoluble debris, the supernatant was used as total protein cell lysate. Twenty-five micrograms of protein were applied to SDS-PAGE gels and immunoblotted as described (36Ramprasad M.P. Li R. Bradley W.A. Gianturco S.H. Human THP-1 monocyte-macrophage membrane binding proteins: distinct receptor(s) for triglyceride-rich lipoproteins.Biochemistry. 1995; 34: 9126-9135Google Scholar). Rabbit polyclonal antibody against human apoB-48R was used as previously described (4Brown M.L. Ramprasad M.P. Umeda P.K. Tanaka A. Kobayashi Y. Watanabe T. Shimoyamada H. Kuo W.L. Li R. Song R. Bradley W.A. Gianturco S.H. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 7488-7493Google Scholar). Mouse anti-actin monoclonal antibody was from Chemicon International, Temecula, CA. Visualization of specific proteins was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Newark, NJ).TRL uptake analysis in THP-1 cellsTHP-1 monocytes were subcultured into 6-well plates and grown in the presence of phorbol ester to induce adherence as previously described (20Gianturco S.H. Ramprasad M.P. Lin A.H. Song R. Bradley W.A. Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.J. Lipid Res. 1994; 35: 1674-1687Google Scholar). After 2 days, cells were washed and incubated with fresh medium containing buffer (control), DMSO (vehicle control), or the PPAR ligands PGJ2 at 10 μM or Wy14643 at 100 μM for 24 h at 37°C. At the end of this incubation period, cells were washed and the test lipoproteins, trypsinized VLDL Sf 100-400, were added at the levels indicated and further incubated for 4 h at 37°C, as previously published (20Gianturco S.H. Ramprasad M.P. Lin A.H. Song R. Bradley W.A. Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.J. Lipid Res. 1994; 35: 1674-1687Google Scholar). Cells were then washed thoroughly with cold PBS to remove lipoproteins and the wells analyzed for TG and protein content as previously described (20Gianturco S.H. Ramprasad M.P. Lin A.H. Song R. Bradley W.A. Cellular binding site and membrane binding proteins for triglyceride-rich lipoproteins in human monocyte-macrophages and THP-1 monocytic cells.J. Lipid Res. 1994; 35: 1674-1687Google Scholar). Duplicate analyses, corrected for no-cell controls, for each lipoprotein concentration were determined. Values from each well differed by <10% and three independent experiments were performed.RESULTSPPARγ activators suppress apoB-48R expression in THP-1 monocytesTo determine whether PPARγ activators regulate apoB-48R expression in human monocytes, we evaluated apoB-48R expression at the mRNA level and protein level in the human monocytic leukemic cell line, THP-1, previously used to characterize, purify, and clone the receptor (4Brown M.L. Ramprasad M.P. Umeda P.K. Tanaka A. Kobayashi Y. Watanabe T. Shimoyamada H. Kuo W.L. Li R. Song R. Bradley W.A. Gianturco S.H. A macrophage receptor for apolipoprotein B48: cloning, expression, and atherosclerosis.Proc. Natl. Acad. Sci. USA. 2000; 97: 7488-7493Google Scholar). THP-1s were incubated with several PPAR activators for up to 24 h prior to analyses. Real-time RT-PCR analysis (Fig. 1)showed that treatment of cells with the PPARγ ligand 15-d-PGJ2 at 10 μM resulted in an ∼95% decrease in mRNA levels of apoB-48R (Fig. 1A). At 20 μM of 15-d-PGJ2, the expression of apoB-48R was almost abolished. At lower 15-d-PGJ2 concentrations, 0.1 and 1.0 μM, the levels of apoB-48R mRNA expression were 60% and 40%, respectively (unpublished observations). The PPARγ activators, troglitazone and pioglitazone, also suppressed the level of apoB-48R mRNA in a dose-dependent manner with ∼50% reduction at 0.1 μM (Fig. 1B, C). Northern blot analysis corroborated the PCR analysis, indicating similar suppression of apoB-48R mRNA at these PPAR activator concentrations (unpublished observations). The time course of mRNA suppression was determined at 100 μM PPAR activators by densitometric analysis of Northern blots and indicated that 15-d-PGJ2 was the most effective agonist, with a reduction in mRNA to 67% at 3 h, 6% at 6 h, and not detectable at 24 h relative to control (100%; relative to GAPDH). Likewise, but later and less effective, troglitazone and pioglitazone showed no lowering of mRNA levels until 6 h with ⩾50% and >90% mRNA reductions at 24 h, respectively. Western blot analysis also showed that 15-d-PGJ2 at a low concentration (10 μM) almost completely abolished apoB-48R protein expression in the same time frame (Fig. 1D). Furthermore, and consistent with the mRNA data, addition of the synthetic PPARγ ligands troglitazone and pioglitazone at 100 μM, suppressed apoB-48R protein expression by >50% (Fig. 1E, F).PPARα activator suppresses apoB-48R expression in THP-1 monocytesWe then examined whether a PPARα activator also regulated apoB-48R expression in THP-1 monocytes at both the mRNA level and the protein level (Fig. 2). Real-time RT-PCR analysis showed that treatment of cells with the PPARα ligand Wy14643 at 25 μM resulted in an ∼40% reduction of apoB-48R mRNA levels and a ∼90% reduction at 50 μM (Fig. 2A). Northern blot analysis showed similar suppression of apoB-48R mRNA by Wy14643 (unpublished observations). Western blot analysis (Fig. 2B), on the other hand, revealed that Wy14643 (50 μM) reduced the expression of apoB-48R protein in THP-1 monocytes by only ∼50% relative to actin, as determined by densitometry, suggesting that the mRNA pool was turning over more rapidly than the apoB-48R protein.Fig. 2The PPARα activator Wy14643 suppresses apoB-48R expression in THP-1 monocytes. A: THP-1 cells were treated with DMSO (control) and Wy14643 at the levels for 24 h, and total RNA was isolated from the cells. Expression of human apoB-48R mRNA was quantified by real-time RT-PCR. Expression of β-actin was determined as an internal control. Values represent expression of apoB-48R mRNA relative to β-actin mRNA at each level of PPAR ligand, with 100% representing the control/vehicle only. B: For Western blotting, total cell protein extracts (25 μg) were analyzed for apoB-48R protein as described in the legend of Fig. 1. Expression of actin was determined as an internal control.View Large Image Figure ViewerDownload (PPT)PPARγ and PPARα activators suppress apoB-48R protein expression in human peripheral blood-borne monocytesTo demonstrate that the regulation of apoB-48R by PPARα and PPARγ activators seen in the human leukemic monocytic cell line THP-1 was more universal and potentially valid in vivo, we also determined and observed similar regulation of apoB-48R protein expression in human native peripheral blood-borne monocytes (Fig. 3). Western blot analysis showed that 15-d-PGJ2 (10 μM) treatment for 24 h almost completely abolished the apoB-48R protein expression in the monocyte-macrophages (Fig. 3A), similar to effects in THP-1 monocyte-macrophages. Troglitazone (50 μM) and pioglitazone (50 μM) suppressed apoB-48R protein expression to a lesser extent, by ∼60% and 40%, respectively, relative to actin controls, as determined by densitometry" @default.
• W2165429105 created "2016-06-24" @default.
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• W2165429105 date "2003-06-01" @default.
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• W2165429105 title "PPARα and PPARγ activators suppress the monocyte-macrophage apoB-48 receptor" @default.
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• W2165429105 doi "https://doi.org/10.1194/jlr.m300077-jlr200" @default.
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