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• W2036702421 abstract "Regulation of expression of the scavenger receptor is thought to play a critical role in the accumulation of lipid by macrophages in atherosclerosis. Tumor necrosis factor-α (TNF-α) has been shown to suppress macrophage scavenger receptor function (van Lenten, B. J., and Fogelman, A. M.(1992) J. Immunol. 148, 112-116). However, the mechanism by which it does so is unknown. We evaluated the mechanism by which TNF-α inhibited macrophage scavenger receptor surface expression and binding of acetylated low density lipoprotein (aLDL). Binding of aLDL to phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages was suppressed by TNF-α in a dose-dependent manner. Inhibition of aLDL binding was paralleled by a reduction of macrophage scavenger receptor protein as detected by the Western blot. TNF-α partially decreased macrophage scavenger receptor mRNA steady state levels in PMA-differentiated THP-1 macrophages, a result that was confirmed by reverse transcription-polymerase chain reaction. PMA increased the luciferase activity driven by the macrophage scavenger receptor promoter in the transfected cells, whereas TNF-α partially reduced luciferase activity. However, macrophage scavenger receptor mRNA half-life was dramatically reduced in cells treated with TNF-α relative to untreated cells. Reduction in macrophage scavenger receptor message in response to TNF-α was dependent on new protein synthesis because it was blocked by cycloheximide. These results indicate that TNF-α regulates macrophage scavenger receptor expression in PMA-differentiated THP-1 macrophages by transcriptional and post-transcriptional mechanisms but principally by destabilization of macrophage scavenger receptor mRNA. Regulation of expression of the scavenger receptor is thought to play a critical role in the accumulation of lipid by macrophages in atherosclerosis. Tumor necrosis factor-α (TNF-α) has been shown to suppress macrophage scavenger receptor function (van Lenten, B. J., and Fogelman, A. M.(1992) J. Immunol. 148, 112-116). However, the mechanism by which it does so is unknown. We evaluated the mechanism by which TNF-α inhibited macrophage scavenger receptor surface expression and binding of acetylated low density lipoprotein (aLDL). Binding of aLDL to phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages was suppressed by TNF-α in a dose-dependent manner. Inhibition of aLDL binding was paralleled by a reduction of macrophage scavenger receptor protein as detected by the Western blot. TNF-α partially decreased macrophage scavenger receptor mRNA steady state levels in PMA-differentiated THP-1 macrophages, a result that was confirmed by reverse transcription-polymerase chain reaction. PMA increased the luciferase activity driven by the macrophage scavenger receptor promoter in the transfected cells, whereas TNF-α partially reduced luciferase activity. However, macrophage scavenger receptor mRNA half-life was dramatically reduced in cells treated with TNF-α relative to untreated cells. Reduction in macrophage scavenger receptor message in response to TNF-α was dependent on new protein synthesis because it was blocked by cycloheximide. These results indicate that TNF-α regulates macrophage scavenger receptor expression in PMA-differentiated THP-1 macrophages by transcriptional and post-transcriptional mechanisms but principally by destabilization of macrophage scavenger receptor mRNA. Macrophage scavenger receptors bind oxidized low density lipoprotein (LDL) ( 1The abbreviations used are: LDLlow density lipoproteinMSRmacrophage scavenger receptoraLDLacetylated LDLLPSlipopolysaccharideTNF-αtumor necrosis factor-αM-CSFmacrophage colony-stimulating factorRTreverse transcriptionPCRpolymerase chain reactionCEcholesteryl esterACATacyl CoA:cholesterol acyltransferasePMAphorbol 12-myristate 13-acetateIFN-μinterferon-μTGF-β1transforming growth factor-β1.) and acetylated LDL (aLDL), as well as other polyanionic ligands, and were initially identified by their ability to bind charge modified LDL but not native LDL(2.Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1904) Google Scholar). Unlike the LDL receptor, expression of the macrophage scavenger receptor is not down-regulated by high levels of intracellular cholesterol. Because of the potential role of this receptor in mediating cholesteryl ester accumulation by macrophages during atherosclerosis, the regulation of expression of these receptors is of considerable important and interest. Circulating monocytes express little or no macrophage scavenger receptor, but receptor mRNA and surface expression are dramatically increased because monocytes differentiate into macrophages in tissue culture. Treatment of monocytes or THP-1 cells (a human monocytic cell line) with phorbol-12 myristate 13-acetate (PMA) also promotes differentiation and induces macrophage scavenger receptor expression(3.Via D.P. Pons L. Dennison D.K. Fanslow A.E. Bernini F. J. Lipid Res. 1989; 30: 1515-1524Abstract Full Text PDF PubMed Google Scholar). Similarly, macrophage colony-stimulating factor (M-CSF) augments macrophage scavenger receptor expression(4.Ishibashi S. Inaba T. Shimano H. Harada K. Inoue I. Mokuno H. Mori N. Gotoha T. Takaku F. Yamada N. J. Biol. Chem. 1990; 265: 14109-14117Abstract Full Text PDF PubMed Google Scholar). low density lipoprotein macrophage scavenger receptor acetylated LDL lipopolysaccharide tumor necrosis factor-α macrophage colony-stimulating factor reverse transcription polymerase chain reaction cholesteryl ester acyl CoA:cholesterol acyltransferase phorbol 12-myristate 13-acetate interferon-μ transforming growth factor-β1. Inhibition of macrophage scavenger receptor expression and activity has been reported in response to interferon-μ (IFN-μ)(5.Fong L. Fong A. Cooper A. J. Biol. Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 6.Geng Y.-J. Hansson G.K. J. Clin. Invest. 1992; 89: 1322-1330Crossref PubMed Scopus (239) Google Scholar), transforming growth factor-β1 (TGF-β1)(7.Bottalico L.A. Wager R.E. Agellon L.B. Assoian R.K. Tabas I. J. Biol. Chem. 1991; 266: 22866-22871Abstract Full Text PDF PubMed Google Scholar), all-trans retinoic acid, dexamethasone(8.Moulton K.S. Wu H. Barnett J. Parthasarathy S. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8102-8106Crossref PubMed Scopus (61) Google Scholar), platelet secretary products(9.Philips D.R. Arnold K. Innerarity T.L. Nature. 1985; 316: 746-748Crossref PubMed Scopus (33) Google Scholar, 10.Aviram M. Metabolism. 1989; 38: 425-430Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 11.Aviram M. Dankner G. Brook J.G. Arteriosclerosis. 1990; 10: 559-563Crossref PubMed Google Scholar), and lymphocyte culture supernatants(12.Fogelman A.M. Seager J. Haberland M.E. Hokom M. Tanaka R. Edward P.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 922-926Crossref PubMed Scopus (36) Google Scholar). Bacterial lipopolysaccharide (LPS), a potent activator of mononuclear phagocytes, can inhibit scavenger receptor activity in human macrophages(13.van Lenten B.J. Fogelman A.M. Seager J. Ribi E. Haberland M. Edwards P. J. Immunol. 1985; 134: 3718-3721PubMed Google Scholar). Most of the inhibitory activity of LPS on the macrophage scavenger receptor could be blocked with an antibody to TNF-α(1.van Lenten B.J. Fogelman A.M. J. Immunol. 1992; 148: 112-116PubMed Google Scholar), suggesting that TNF-α, which is synthesized in response to LPS, mediated the LPS effect. However, the molecular mechanism(s) by which TNF-α inhibits macrophage scavenger receptor activity have not been elucidated. In this study, we demonstrate that TNF-α inhibits macrophage scavenger receptor binding activity, surface protein expression, and mRNA levels, with subsequent down-regulation of macrophage scavenger receptor-mediated ACAT activity and cholesterol esterification in PMA-differentiated THP-1 macrophages. Although macrophage scavenger receptor transcriptional activity was modestly reduced in response to TNF-α, in the presence of actinomycin D, macrophage scavenger receptor mRNA half-life was significantly reduced, implying that TNF-α inhibits macrophage scavenger receptor expression principally by post-transcriptional decreases in macrophage scavenger receptor mRNA stability. Disposable tissue culture materials were purchased from Corning Glass Works (Corning, NY). Medium RPMI 1640 medium, L-glutamine, penicillin, streptomycin, and fetal calf serum were purchased from Life Technologies, Inc. A 100-base pair DNA ladder was purchased from Life Technologies, Inc., MD. 125I was obtained from ICN Biochemicals (Costa Mesa, CA). EGTA, leupeptin, aprotinin and DNA molecular weight marker V were obtained from Boehringer Mannheim. Sodium orthovanadate was obtained from Aldrich. HEPES, NaCl, glycerol, Triton X-100, MgCl2, phenylmethylsulfonyl fluoride, PMA, actinomycin D, cycloheximide, and bovine serum albumin (Fraction V) were purchased from Sigma. Immobilon membrane was purchased from Millipore, MA. DuPont Western blot Chemiluminescence Reagent, Renaissance®, a nonradioactive light-emitting system, was purchased from DuPont NEN. Human recombinant TNF-α and human recombinant M-CSF were obtained from R & D Systems (Minneapolis, MN). Rabbit polyclonal anti-human macrophage scavenger receptor antibody, i.e. hSRI-2 anti-macrophage scavenger receptor peptide antibody, was a gift from Dr. T. Kodama (University of Tokyo, Tokyo, Japan)(14.Naito M. Suzuki H. Mori T. Matsumoto A. Kodama T. Takahashi K. Am. J. Pathol. 1992; 141: 591-599PubMed Google Scholar). Donkey anti-rabbit IgG-horseradish peroxidase conjugate was obtained from Boehringer Mannheim. The human monocytic macrophage THP-1 cell line (15.Tsuchiya S. Yamabe M. Yamaguchi Y. Kobayashi Y. Konno T. Tada K. Int. J. Cancer. 1980; 26: 171-176Crossref PubMed Scopus (1620) Google Scholar, 16.Tsuchiya S. Kobayashi Y. Goto Y. Okumura H. Nakae S. Konno T. Tada K. Cancer Res. 1982; 42: 1530-1536PubMed Google Scholar) was purchased from ATCC (Rockville, MD) and followed the ATCC protocol to grow. Cells were incubated in 5% CO2 in air at 37°C. In this paper, human suspension monocytic THP-1 cell is referred to as THP-1 monocyte; for PMA-differentiated adhesion macrophage-like THP-1 cell is referred to as PMA-differentiated THP-1 macrophage or THP-1 macrophage as described(7.Bottalico L.A. Wager R.E. Agellon L.B. Assoian R.K. Tabas I. J. Biol. Chem. 1991; 266: 22866-22871Abstract Full Text PDF PubMed Google Scholar, 16.Tsuchiya S. Kobayashi Y. Goto Y. Okumura H. Nakae S. Konno T. Tada K. Cancer Res. 1982; 42: 1530-1536PubMed Google Scholar, 17.Hara H. Tanishita H. Yokoyama S. Tajima S. Yamamoto A. Biochem. Biophys. Res. Commun. 1987; 146: 802-808Crossref PubMed Scopus (60) Google Scholar, 18.Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (829) Google Scholar). Human LDL (d = 1.019-1.063 gm/ml) was prepared as described(19.Hajjar D.P. Falcone D.J. Fabricant C.G. Fabricant J. J. Biol. Chem. 1985; 260: 6124-6128Abstract Full Text PDF PubMed Google Scholar). All LDL preparations were screened for peroxides(20.Marshall P. Warso M. Lands W. Anal. Biochem. 1985; 145: 192-199Crossref PubMed Scopus (139) Google Scholar). LDL was labeled by the method of Bilheimer et al.(21.Bilheimer D. Eisenberg S. Levy R. Biochim. Biophys. Acta. 1972; 260: 212-221Crossref PubMed Scopus (1184) Google Scholar) as described by Goldstein et al.(22.Goldstein J. Basu S. Brown M. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1274) Google Scholar) yielding 125I-aLDL with specific activity between 120 and 220 cpm/ng protein. The binding of 125I-aLDL was performed according to published methods(22.Goldstein J. Basu S. Brown M. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1274) Google Scholar). 125I-aLDL was added to cells in the presence or the absence of a 100-fold excess of unlabeled aLDL at 4°C. After 2 h on a rotary shaker, the cells were washed three times with ice-cold phosphate-buffered saline containing 2 mg/ml of bovine serum albumin and then washed two times with ice-cold phosphate-buffered saline. The cells were solubilized in 0.2 M NaOH, and radioactivity was quantified in a Searle 1185 gamma counter (Searle Radiographics, Inc., IL). Nonspecifically bound 125I-aLDL was determined by incubating cells with a 100-fold excess of unlabeled aLDL. Specific aLDL binding was determined by subtracting nonspecifically bound counts from total counts. Binding was normalized to protein content of the cell layer. Total RNA was isolated by the guanidium isothiocyanate method(23.Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5894-5898Crossref Scopus (16608) Google Scholar). Northern blot analyses were performed as described(24.Davis L. Dibner M. Battery J. Basic Methods in Molecular Biology. Elsevier Science Publishing Co., Inc., New York1986: 130-146Crossref Google Scholar, 25.Hsu H.-Y. Nicholson A.C. Hajjar D.P. J. Biol. Chem. 1994; 269: 9213-9220Abstract Full Text PDF PubMed Google Scholar). The cDNA for the human macrophage scavenger receptor was a gift from Dr. T. Kodama (University of Tokyo, Tokyo, Japan)(26.Matsumoto A. Naito M. Itakura H. Ikemoto S. Asaoka H. Hayakawa I. Kanamori H. Aburatani H. Takaku F. Suzuki H. Kobari Y. Miyai T. Takahashi K. Cohen E.H. Wydro R. Housman D.E. Kodama T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9133-9137Crossref PubMed Scopus (300) Google Scholar). The cDNA for human 28 S ribosomal RNA was a gift from Dr. Iris L. Gonzalez (Hahnemann University, Philadelphia, PA). RNA from Northern blots was quantified using a PhosphorImager® (Molecular Dynamics, Sunnyvale, CA), and normalized by comparison with mRNA of 28 S ribosomal RNA, a constitutively expressed gene. -Cells were subpassaged the day before transfection and replaced with fresh medium. The transfection method is described elsewhere(27.Dorsett D.L. Keshet I. Winocour E. J. Virol. 1983; 48: 218-228Crossref PubMed Google Scholar). Briefly, the plasmid of macrophage scavenger receptor promoter in luciferase reporter gene, such as plasmids HACLDL Xba-A1-luc promoter and HACLDL Xba-A1-luc Enhancer, respectively (a gift from Dr. C. Glass, University of California, San Diego, CA)(28.Wu H. Moulton K. Horvai A. Parik S. Glass C.K. Mol. Cell. Biol. 1994; 14: 2129-2139Crossref PubMed Google Scholar), were co-transfected with β-galactosidase as to monitor the transfection efficiency. Transfected cells were treated with various reagents as indicated, and luciferase activities were measured according to the method previously described(25.Hsu H.-Y. Nicholson A.C. Hajjar D.P. J. Biol. Chem. 1994; 269: 9213-9220Abstract Full Text PDF PubMed Google Scholar). β-Galactosidase assay was followed the protocol supplied by Promega Co. Acid (lysosomal) CE hydrolase activity was assayed at pH 3.9(29.Haley N.J. Fowler S. deDuve C. J. Lipid Res. 1980; 21: 961-969Abstract Full Text PDF PubMed Google Scholar), and, neutral (cytoplasmic) CE hydrolase activity was assayed at pH 7.0(30.Hajjar D.P. Weksler B. J. Lipid Res. 1983; 24: 1176-1185Abstract Full Text PDF PubMed Google Scholar). ACAT activity in homogenates was measured as an index of CE synthetic activity by determining the rate of CE synthesis from [1-14C]oleoyl CoA using exogenous free cholesterol incorporated into unilamellar liposomes containing egg phosphatidylcholine(31.Hajjar D.P. Weksler B.B. Falcone D.J. Hefton J.H. Tack-Goldman K. Minick C.R. J. Clin. Invest. 1982; 70: 479-488Crossref PubMed Scopus (105) Google Scholar). THP-1 cells were exposed to a [3H]oleic acid (sp. act. 40,000 dpm/nmol)/albumin mixture (final concentration, 100 μM oleate, 20 μM albumin in the absence of fetal calf serum) for 24 h at 37°C. Cell lipids were extracted, and radioactivity in CE was assessed after separation by TLC as described(32.Hajjar D.P. Wight T. Smith S. Am. J. Pathol. 1980; 100: 683-705PubMed Google Scholar). The method for immunoblotting is described elsewhere(33.Margolis B. Rhee S.G. Felder S. Mervic M. Lyall R. Levitzki A. Ullrich A. Zilberstein A. Schlessinger J. Cell. 1989; 57: 1101-1107Abstract Full Text PDF PubMed Scopus (510) Google Scholar, 34.Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (204800) Google Scholar). Briefly, THP-1 monocytes treated with PMA (150 nM), or PMA (150 nM) + TNF-α (200 units/ml) for 24 h. Whole cell lysates (120 μg of protein) were solubilized in SDS sample buffer with mild reducing conditions (boiling for 5 min in the presence of 2-mercaptoethanol). Protein was loaded in each lane, separated by SDS-polyacrylamide gel electrophoresis, and transferred to an Immobilon membrane. The presence of macrophage scavenger receptor protein was examined by incubation the membrane with hSRI-2 anti-macrophage scavenger receptor peptide antibody (a rabbit polyclonal anti-human macrophage scavenger receptor antibody), which recognizes the collagen-like domain that is shared between types SR-AI and SR-AII macrophage scavenger receptor proteins(14.Naito M. Suzuki H. Mori T. Matsumoto A. Kodama T. Takahashi K. Am. J. Pathol. 1992; 141: 591-599PubMed Google Scholar). Blots were stained with a donkey anti-rabbit IgG-horseradish peroxidase conjugate. Protein visualization was performed with Renaissance®, DuPont Western blot Chemiluminescence Reagent, (DuPont NEN). Manufacturer-provided protocols were followed. The reagents in RNA PCR kit for RT-PCR amplification were purchased from Perkin-Elmer. The oligonucleotides for the primers (26.Matsumoto A. Naito M. Itakura H. Ikemoto S. Asaoka H. Hayakawa I. Kanamori H. Aburatani H. Takaku F. Suzuki H. Kobari Y. Miyai T. Takahashi K. Cohen E.H. Wydro R. Housman D.E. Kodama T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9133-9137Crossref PubMed Scopus (300) Google Scholar, 35.Geng Y.-J. Kodama T. Hansson G.K. Arterioscler. Thromb. 1994; 14: 798-806Crossref PubMed Google Scholar) and the procedures for RT-PCR amplification were as described (35.Geng Y.-J. Kodama T. Hansson G.K. Arterioscler. Thromb. 1994; 14: 798-806Crossref PubMed Google Scholar) with some modification. After electrophoresis on 2% agarose gels, the PCR products were stained with ethidium bromide and visualized on a UV transilluminator. Quantitative analysis of PCR products was performed by using Southern blot, and the membranes were hybridized with the 32P-labeled internal probe as described (35.Geng Y.-J. Kodama T. Hansson G.K. Arterioscler. Thromb. 1994; 14: 798-806Crossref PubMed Google Scholar) and quantified using a PhosphorImager®. Differences in intensity of PCR product bands were confirmed by 10-fold serial dilution of cDNA samples to ensure comparability and that the plateau of amplification had not been reached as described(36.Villiers W.J.S.D. Fraser I.P. Hughes D.A. Doyle A.G. Gordon S. J. Exp. Med. 1994; 180: 705-709Crossref PubMed Scopus (124) Google Scholar). Protein amounts were determined by the method of Lowry et al.(37.Lowry O. Rosebrough N. Farr A. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or the Bio-Rad protein assay. Statistical differences between the experimental groups were examined by analysis of variance, and statistical significance was determined at a p level of <0.05. All data are expressed as the means ± S.E. PMA and M-CSF equivalently increased the specific binding (4°C) of 125I-aLDL to THP-1 macrophages compared with untreated THP-1 monocytes (Fig. 1A). Co-treatment of PMA- or M-CSF-differentiated THP-1 cells with TNF-α reduced specific binding of 125I-aLDL to baseline (control) levels (Fig. 1A) in a dose-dependent manner (Fig. 2). The effect of TNF-α on 125I-aLDL binding resulted from a decrease in the number of binding sites (Bmax) without significant change in receptor affinity (Kd). Scatchard analysis (Fig. 1B) demonstrated that PMA or M-CSF increased Bmax by 6- or 7-fold, respectively, as compared with undifferentiated THP-1 monocytes (undifferentiated THP-1 monocytes (control) = 1.66 × 105 sites/cell; PMA-differentiated THP-1 macrophages) = 9.71 × 105 sites/cell; M-CSF treated THP-1 cells = 10.6 × 105 sites/cell). The Bmax of undifferentiated THP-1 monocytes (control), PMA-differentiated THP-1 macrophages treated with TNF-α (PMA + TNF-α), as well as THP-1 monocytes treated with TNF-α and M-CSF-differentiated THP-1 macrophages treated with TNF-α, (data not shown) were equivalent (each approximately 1.23-1.66 × 105 sites/cell). Kd values were not significantly altered by any treatment (Fig. 1B).Figure 2:Dose response effect of TNF-α on aLDL binding in PMA-differentiated THP-1 macrophages. PMA-differentiated THP-1 macrophages were incubated with varying concentrations of TNF-α (1-300 units/ml). Specific aLDL binding (125I-aLDL, 20 μg/ml) was performed as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Western analysis (Fig. 3) revealed that little or no macrophage scavenger receptor protein was expressed in THP-1 monocytes. PMA-differentiated THP-1 macrophages expressed scavenger receptor protein, but TNF-α suppressed the expression of macrophage scavenger receptor protein when THP-1 cells co-incubated with PMA. TNF-α did not cause any signs of general cellular toxicity. Cells treated with PMA/TNF-α remained adherent and morphologically similar to PMA-treated cells (THP-1 macrophages). Furthermore, there was no decrease in protein content or cell number or any increase in the uptake of trypan blue (data not shown). To dissect the molecular mechanisms by which TNF-α suppressed macrophage scavenger receptor protein expression, we initially examined the influence of TNF-α on macrophage scavenger receptor mRNA levels. Northern blot analyses of PMA-differentiated THP-1 macrophages revealed a time-dependent increase in macrophage scavenger receptor mRNA steady state levels in response to PMA (Fig. 4, A and B). Expression peaked at 48 h. Macrophage scavenger receptor mRNA was not detected in undifferentiated THP-1 monocytes (not treated with PMA), and TNF-α had no effect on this baseline expression. TNF-α decreased macrophage scavenger receptor mRNA expression by 80 (at 20 h) and 70% (at 48 h), respectively, in PMA-differentiated cells relative to PMA-differentiated cells not treated with TNF-α (THP-1 macrophages) (Fig. 4, A and B). RT-PCR demonstrated that TNF-α decreased both human macrophage scavenger receptor types SR-AI and SR-AII mRNA in PMA-treated THP-1 macrophages (Fig. 4C). Dose response experiments showed that maximal inhibition of macrophage scavenger receptor mRNA levels (similar to macrophage scavenger receptor surface expression as determined by aLDL binding) by TNF-α occurred at 200 units/ml (data not shown). To examine if decreased macrophage scavenger receptor mRNA expression in response to TNF-α in PMA-differentiated THP-1 macrophages resulted from decreased transcription of the macrophage scavenger receptor gene, assays for luciferase activity driven by macrophage scavenger receptor gene promoter were performed. THP-1 cells were transiently transfected by the DEAE-dextran sulfate method with plasmid MSR-Luc luciferase construct consisting of 5′ upstream sequences of the macrophage scavenger receptor promoter region. Luciferase activity was measured in untreated cells, cells treated with PMA, and cells treated with PMA + TNF-α (200 units/ml). PMA induced luciferase activity driven by the macrophage scavenger receptor gene promoter by 3.5-fold. TNF-α reduced luciferase activity by 20% after 12 h (Fig. 5). To determine if TNF-α reduced the level of macrophage scavenger receptor mRNA by increasing its rate of degradation, we measured the half-life of macrophage scavenger receptor mRNA in the presence of the transcription inhibitor actinomycin D (5 μg/ml). TNF-α shortened the half-life of macrophage scavenger receptor mRNA from 40 ± 3 to 10 ± 2 h (Fig. 6, A, B, and C). Therefore, the reduction in macrophage scavenger receptor message by TNF-α appears to be mediated principally by a post-transcriptional mechanism, namely, accelerating the degradation of macrophage scavenger receptor mRNA. Moreover, incubation of cells with the protein synthesis inhibitor cycloheximide (10 μg/ml) prevented the decrease of macrophage scavenger receptor mRNA at both 3 and 6 h in TNF-α-treated samples (Fig. 7). These findings suggest that the effect(s) of TNF-α on macrophage scavenger receptor mRNA destabilization requires new protein synthesis.Figure 7:Northern blots showing reversal by cycloheximide of the destabilizing effects of TNF-α on MSR mRNA. PMA-differentiated THP-1 macrophages were treated with cycloheximide (10 μg/ml) for 1 h before the addition of TNF-α (200 units/ml). PMA-differentiated THP-1 macrophages (PMA (Control)), PMA-differentiated THP-1 macrophages treated with TNF-α (PMA + TNF-α), and PMA-differentiated THP-1 macrophages treated with TNF-α and incubated with cycloheximide (PMA + TNF-α + CHX) were harvested 3 and 6 h after exposure to TNF-α. Northern blots were hybridized with 32P-labeled cDNAs of MSR and 28 S ribosomal RNA, quantitated by PhosphorImager®, and normalized by comparison with 28 S. All of the data are expressed as a percentage relative to THP-1 macrophages (t = 3 h). The relative intensity of MSR mRNA is plotted against time. Pretreating THP-1 macrophages for 1 h with cycloheximide had no effect on the basal level of MSR mRNA (data not shown). Similar results were obtained in three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lastly, we characterized the role of TNF-α in macrophage scavenger receptor-mediated CE metabolism by evaluating parameters of the CE cycle. ACAT activity (Fig. 8A) as well as esterification of free cholesterol with [3H]oleic acid to CE (Fig. 8B) were evaluated in PMA-differentiated THP-1 macrophages and PMA-differentiated THP-1 cells treated with TNF-α. TNF-α caused a significant (65%) reduction in ACAT activity and a similar decrease in the synthesis of nascent CE from free fatty acids as compared with untreated cells. These decreases paralleled the decrease in aLDL binding and most likely reflect a decrease in cholesterol delivery. Finally, the effects of TNF-α on CE hydrolase activities were measured, but no significant difference in acid CE hydrolase or neutral CE hydrolase activities in response to TNF-α was observed (data not shown). Expression of the macrophage scavenger receptor (acetylated LDL receptor) occurs as monocytes differentiate into macrophages in tissue culture over a period of several days, a process that mimics what is thought to occur as blood monocytes enter tissue to become tissue macrophages. These differentiation events can be mimicked by treating monocytes or some (but not all) monocytic cell lines, such as THP-1 cells, with PMA. PMA activates protein kinase C (8.Moulton K.S. Wu H. Barnett J. Parthasarathy S. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8102-8106Crossref PubMed Scopus (61) Google Scholar, 38.Akeson A.L. Schroeder K. Woods C. Schmidt C.J. Jones W.D. J. Lipid Res. 1991; 32: 1699-1707Abstract Full Text PDF PubMed Google Scholar) in THP-1 cells concomitantly with the differentiation of this cell type into macrophage-like cells(7.Bottalico L.A. Wager R.E. Agellon L.B. Assoian R.K. Tabas I. J. Biol. Chem. 1991; 266: 22866-22871Abstract Full Text PDF PubMed Google Scholar, 17.Hara H. Tanishita H. Yokoyama S. Tajima S. Yamamoto A. Biochem. Biophys. Res. Commun. 1987; 146: 802-808Crossref PubMed Scopus (60) Google Scholar, 18.Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (829) Google Scholar). Fogelman and his colleagues had previously shown that LPS inhibited scavenger receptor activity in human macrophages (13.van Lenten B.J. Fogelman A.M. Seager J. Ribi E. Haberland M. Edwards P. J. Immunol. 1985; 134: 3718-3721PubMed Google Scholar) and that this inhibitory effect could be blocked with an antibody to TNF-α(1.van Lenten B.J. Fogelman A.M. J. Immunol. 1992; 148: 112-116PubMed Google Scholar). We utilized PMA-differentiated THP-1 macrophages to evaluate the molecular mechanisms by which TNF-α inhibited macrophage scavenger receptor expression. The inhibitory effect of TNF-α on macrophage scavenger receptor expression and activity is comparable with previously demonstrated inhibitory effects of IFN-μ and TGF-β1 on the macrophage scavenger receptor. TNF-α inhibited macrophage scavenger receptor activity (aLDL binding, Fig. 1), protein synthesis (Fig. 3), and mRNA steady state levels (Fig. 4). TNF-α caused a 6-fold decrease in macrophage scavenger receptor number without affecting receptor affinity in PMA-differentiated THP-1 macrophages. IFN-μ inhibited macrophage scavenger receptor activity in human (5.Fong L. Fong A. Cooper A. J. Biol. Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar) or mouse monocyte-derived macrophages(6.Geng Y.-J. Hansson G.K. J. Clin. Invest. 1992; 89: 1322-1330Crossref PubMed Scopus (239) Google Scholar). IFN-μ inhibited binding, internalization, and degradation of aLDL and reduced macrophage scavenger receptor mRNA steady state levels in comparison with untreated cells. This resulted in a reduction of cholesterol and CE content. Inhibition of binding resulted from decreased numbers of aLDL binding sites without a significant change in receptor affinity. Similarly, TGF-β1 inhibited macrophage scavenger receptor activity and mRNA in PMA-differentiated THP-1 macrophages (7.Bottalico L.A. Wager R.E. Agellon L.B. Assoian R.K. Tabas I. J. Biol. Chem. 1991; 266: 22866-22871Abstract Full Text PDF PubMed Google Scholar) and caused a decrease in binding of aLDL, degradation of aLDL, and ACAT activity relative to PMA-differentiated THP-1 cells. TGF-β1 caused a 2-fold decrease in macrophage scavenger receptor number as well as a decrease in receptor affinity. Inhibition of macrophage scavenger receptor expression in response to cytokines is cell-specific. TNF-α and IFN-μ increased scavenger receptor activity and mRNA in rabbit vascular smooth muscle cells (39.Li H.-M. Freeman M. Libby P. J. Clin. Invest. 1995; 95: 122-133Crossref PubMed Google Scholar). TGF-β, in combination with other cytokines, increased scavenger receptor activity in rabbit smooth muscle cells(40.Gong Q. Pitas R.E. J. Biol. Chem. 1995; 270: 21672-21678Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The mechanism for the divergent effects of these cytokines on these two cell types (macrophages and smooth muscle cells) is unknown. However, it may reflect the fact that basal level of scavenger receptor expression in these two cell types is markedly different. Macrophages constitutively express macrophage scavenger receptor, whereas smooth muscle cells express little or no scavenger receptor mRNA or protein unless induced by cytokines, growth factors, or PMA(39.Li H.-M. Freeman M. Libby P. J. Clin. Invest. 1995; 95: 122-133Crossref PubMed Google Scholar, 40.Gong Q. Pitas R.E. J. Biol. Chem. 1995; 270: 21672-21678Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). ( 2H.-Y. Hsu, A. C. Nicholson, and D. P. Hajjar, unpublished data.) Macrophage scavenger receptor mRNA steady state levels in THP-1 monocytes were up-regulated by the addition of PMA; however, TNF-α decreased macrophage scavenger receptor mRNA steady state levels to about 30% of THP-1 macrophages (Fig. 4, A and B). Two isoforms of macrophage scavenger receptor, types SR-AI and SR-AII, on human macrophages (including PMA-differentiated THP-1 macrophages) are encoded by a single gene that gives rise to an alternatively spliced primary transcript(41.Freeman M. Ashkenas J. Rees D.J. Kingsley D.M. Copeland N.G. Jenkins N.A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8810-8814Crossref PubMed Scopus (256) Google Scholar, 42.Emi M. Asaoka A. Matsumoto A. Itakura H. Kurihara Y. Wada Y. Kanamori H. Yazaki Y. Takahashi E. Lepert M. Lalouel J.-M. Kodama T. Mukai T. J. Biol. Chem. 1993; 268: 2120-2125Abstract Full Text PDF PubMed Google Scholar, 43.Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Crossref PubMed Scopus (1050) Google Scholar). To further determine the effect of TNF-α on the differential expression of the macrophage scavenger receptor gene subtypes, RT-PCR amplification for human macrophage scavenger receptor types SR-AI and SR-AII in PMA-differentiated THP-1 macrophages was performed. We found that TNF-α destabilizes the expression of both macrophage scavenger receptor isoforms (SR-AI and SR-AII) during the differentiation of THP-1 monocytes to THP-1 macrophages (Fig. 4C). Glass and his colleagues have studied transcriptional regulation of the macrophage scavenger receptor gene in PMA-differentiated THP-1 macrophages(8.Moulton K.S. Wu H. Barnett J. Parthasarathy S. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8102-8106Crossref PubMed Scopus (61) Google Scholar, 28.Wu H. Moulton K. Horvai A. Parik S. Glass C.K. Mol. Cell. Biol. 1994; 14: 2129-2139Crossref PubMed Google Scholar, 44.Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell. Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (159) Google Scholar). They have identified transcription factor binding sites for AP-1, SP-1, and ets in the promoter of macrophage scavenger receptor gene from THP-1 macrophages(8.Moulton K.S. Wu H. Barnett J. Parthasarathy S. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8102-8106Crossref PubMed Scopus (61) Google Scholar). Furthermore, the mechanisms for developmental regulation and cell-specific expression of macrophage scavenger receptor gene have been investigated. Complicated growth- and differentiation-related regulatory pathways of macrophage scavenger receptor gene transcription in THP-1 macrophages have been proposed(28.Wu H. Moulton K. Horvai A. Parik S. Glass C.K. Mol. Cell. Biol. 1994; 14: 2129-2139Crossref PubMed Google Scholar, 44.Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell. Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (159) Google Scholar). Specifically, positive transcriptional control of the macrophage scavenger receptor is dependent on the combinatorial interactions of multiple positive factors (including Spi-1/PU.1, which binds to the region I, and a ternary complex of c-Jun, JunB, and an ets2-like protein, which binds to the region IV) and negative factors (undefined inhibitory elements, which bind to the regions II, III and VI)(44.Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell. Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (159) Google Scholar). Moreover, the ternary complex binding to the region IV is a target for transcriptional activation following stimulation of THP-1 monocytes with PMA. Decreased expression of macrophage scavenger receptor mRNA in response to TNF-α resulted, in part, from decreased transcriptional activity of macrophage scavenger receptor gene. Specifically, PMA increased macrophage scavenger receptor mRNA in THP-1 monocytes via the transcriptional activation of the macrophage scavenger receptor gene as shown (Fig. 5) and as described(44.Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell. Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (159) Google Scholar). TNF-α reduced macrophage scavenger receptor gene transcription by 20% (Fig. 5). TNF-α has been show to decrease the transcriptional rate of many genes(45.Solis-Herruzo J.A. Brenner D.A. Chojkier M. J. Biol. Chem. 1988; 263: 5841-5845Abstract Full Text PDF PubMed Google Scholar, 46.Conway E.M. Rosenberg R.D. Mol. Cell. Biol. 1988; 8: 5588-5592Crossref PubMed Scopus (325) Google Scholar, 47.Pape M.E. Kim K.-H. Mol. Cell. Biol. 1989; 9: 974-982Crossref PubMed Scopus (29) Google Scholar). However, other genes are induced in response to TNF-α mediated by the induction of transcription factors such as NFκB, AP-1, IRF-1, and NF-GMa(48.Schutze S. Machleidt T. Kronke M. Semin. Oncol. 1992; 19: 16-24PubMed Google Scholar). Whether the effect of TNF-α on transcriptional down-regulation of macrophage scavenger receptor mRNA alters the combinatorial interactions of positive and negative factors as described (44.Moulton K.S. Semple K. Wu H. Glass C.K. Mol. Cell. Biol. 1994; 14: 4408-4418Crossref PubMed Scopus (159) Google Scholar) remains to be further investigated. Our results demonstrate that in the presence of actinomycin D (an inhibitor of transcription), TNF-α inhibited macrophage scavenger receptor mRNA principally by reducing macrophage scavenger receptor mRNA half-life (Fig. 6). Although both IFN-μ (5.Fong L. Fong A. Cooper A. J. Biol. Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 6.Geng Y.-J. Hansson G.K. J. Clin. Invest. 1992; 89: 1322-1330Crossref PubMed Scopus (239) Google Scholar) and TGF-β1 (7.Bottalico L.A. Wager R.E. Agellon L.B. Assoian R.K. Tabas I. J. Biol. Chem. 1991; 266: 22866-22871Abstract Full Text PDF PubMed Google Scholar) inhibited macrophage scavenger receptor mRNA steady state levels, it was undetermined if these cytokines reduced macrophage scavenger receptor transcription or if the reduction in mRNA steady state levels was due to increased mRNA degradation. The TNF-α reduction in macrophage scavenger receptor mRNA half-life was inhibited by cycloheximide, implying that new protein synthesis was necessary and suggesting that TNF-α induced expression of protein(s) that accelerated macrophage scavenger receptor mRNA degradation (Fig. 7). A reduction of endothelial cell nitric oxide synthase mRNA half-life has also been demonstrated in response to TNF-α (49.Yoshizumi Y. Perrella M.A. Burnett J.C. Lee M.-E. Circ. Res. 1993; 73: 205-209Crossref PubMed Scopus (690) Google Scholar, 50.Mohamed F. Monge J.C. Gordan A. Cernacek P. Blais D. Stewart D.J. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 52-57Crossref PubMed Scopus (74) Google Scholar, 51.Mohamed F. Monge J.C. Lukose A. Farshadi F. Cernacek P. Stewart D.J. FASEB J. 1995; 9 (Abstr. 1890): 326Google Scholar) and was dependent on protein synthesis(49.Yoshizumi Y. Perrella M.A. Burnett J.C. Lee M.-E. Circ. Res. 1993; 73: 205-209Crossref PubMed Scopus (690) Google Scholar). To assess the effects of TNF-α on cholesterol trafficking in THP-1 macrophages, the role of TNF-α in macrophage scavenger receptor-mediated CE metabolism was characterized by evaluating parameters in the CE cycle. TNF-α decreased ACAT activity (Fig. 8A) and esterification of free cholesterol (Fig. 8B) by 65% as compared with untreated cells and paralleled the decrease in aLDL binding. Decreased ACAT activity most likely resulted from decreased substrate (e.g. cholesterol) availability to the enzyme. However, we cannot rule out the possibility that decreased ACAT activity in response to TNF-α may occur through phosphorylation of ACAT by protein kinase(52.Corton J.M. Hardie D.G. Eur. J. Biochem. 1992; 204: 203-208Crossref PubMed Scopus (15) Google Scholar). CE hydrolytic enzymes such as acid CE hydrolase and neutral CE hydrolase activities were unaffected by TNF-α treatment. These findings may have physiologic significance in the pathogenesis of atherosclerosis. Scavenger receptors are expressed by macrophages in atherosclerotic lesions and are believed to mediate the binding and uptake of modified LDL including oxidized LDL. Cytokines produced by cells comprising the atheroma (macrophages, endothelial cells, smooth muscle cells, and lymphocytes) modulate macrophage scavenger receptor expression in vitro and are thought to participate in modulating expression in vivo. The expression of TNF-α is increased in atherosclerotic tissue (53.Rus H.G. Niculescu F. Vlaicu R. Atherosclerosis. 1991; 89: 247-254Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 54.Tipping P.G. Hancock W.W. Am. J. Pathol. 1993; 142: 1721-1728PubMed Google Scholar) in comparison with normal vascular tissue. TNF-α within the atherosclerotic lesion could have a modulatory or inhibitory effect on macrophage scavenger receptor-mediated accumulation of oxidized lipoprotein by macrophages. We have previously demonstrated that TNF-α can up-regulate expression of the LDL receptor by HepG2 cells (55.Stopeck A.T. Nicholson A.C. Mancini F.P. Hajjar D.P. J. Biol. Chem. 1993; 268: 17489-17494Abstract Full Text PDF PubMed Google Scholar) by increasing LDL receptor gene transcription. This study and our previous work (55.Stopeck A.T. Nicholson A.C. Mancini F.P. Hajjar D.P. J. Biol. Chem. 1993; 268: 17489-17494Abstract Full Text PDF PubMed Google Scholar) demonstrate how cytokines can modulate transcriptional and post-transcriptional regulation of lipoprotein receptors. These regulatory effects may potentially alter lipoprotein metabolism in vascular and hepatic tissues and alter pathophysiologic processes. We thank Terrance Win for technical assistance." @default.
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