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• W2019796869 abstract "Macrophage-derived apoE has been shown to play an important role in the susceptibility of the vessel wall to atherosclerosis. Previous studies have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene. The current studies were undertaken to evaluate the existence of additional post-transcriptional regulatory loci for the effect of sterols on apoE synthesis and secretion. Using a macrophage cell line transfected to constitutively express an apoE cDNA to facilitate detection of a post-transcriptional regulatory locus, we demonstrated that preincubations in 25-hydroxycholesterol and cholesterol lead to increased apoE secretion in pulse/chase experiments. Examination of cell lysates in these experiments showed that apoE not secreted by control cells was degraded and not detectable, suggesting that the preincubation in sterols increased secretion by decreasing degradation of newly synthesized apoE. The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degraded significant amounts of newly synthesized total protein and newly synthesized apoE. In this fraction, degradation of total protein and apoE was unaffected by chloroquine but was substantially reduced by N-acetyl-Leu-Leu-norleucinal plusN-acetyl-Leu-Leu-methioninal or by lactacystin, suggesting the involvement of proteasomes. Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradation. Similar results were obtained using intermediate density fractions isolated from human monocyte-derived macrophages. The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment, which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradation in this fraction. Inhibition of the degradation of apoE in this fraction likely contributes to the increased apoE secretion observed in sterol-enriched cells. Macrophage-derived apoE has been shown to play an important role in the susceptibility of the vessel wall to atherosclerosis. Previous studies have shown that macrophage sterol content modulates apoE synthesis and secretion, associated with a large transcriptional response of the apoE gene. The current studies were undertaken to evaluate the existence of additional post-transcriptional regulatory loci for the effect of sterols on apoE synthesis and secretion. Using a macrophage cell line transfected to constitutively express an apoE cDNA to facilitate detection of a post-transcriptional regulatory locus, we demonstrated that preincubations in 25-hydroxycholesterol and cholesterol lead to increased apoE secretion in pulse/chase experiments. Examination of cell lysates in these experiments showed that apoE not secreted by control cells was degraded and not detectable, suggesting that the preincubation in sterols increased secretion by decreasing degradation of newly synthesized apoE. The measurement of total protein and apoE degradation in cell fractions revealed an intermediate density fraction that degraded significant amounts of newly synthesized total protein and newly synthesized apoE. In this fraction, degradation of total protein and apoE was unaffected by chloroquine but was substantially reduced by N-acetyl-Leu-Leu-norleucinal plusN-acetyl-Leu-Leu-methioninal or by lactacystin, suggesting the involvement of proteasomes. Preincubation in sterol/oxysterol or acetylated low density lipoprotein did not modify total protein degradation by this fraction but inhibited apoE degradation. Similar results were obtained using intermediate density fractions isolated from human monocyte-derived macrophages. The results of our studies indicate that newly synthesized apoE in the macrophage can be degraded in an intermediate density nonlysosomal cellular compartment, which is sensitive to proteasomal inhibitors. Alteration of cellular lipid homeostasis by preincubation in sterol/oxysterol or acetylated low density lipoprotein inhibits apoE, but not total protein, degradation in this fraction. Inhibition of the degradation of apoE in this fraction likely contributes to the increased apoE secretion observed in sterol-enriched cells. Apolipoprotein E is synthesized by hepatic cells and a large number of extrahepatic cell types (1Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 2Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). In the liver, apoE is produced as a surface constituent of lipoprotein particles and serves as a ligand for several cell membrane endocytic receptors found in liver and peripheral cells. In addition, apoE is a highly charged protein and has strong affinity for negatively charged proteoglycans, including those found on the cell surface. Consistent with this, apoE has been detected in a cell surface pool in hepatic cells and macrophages, where it may be involved in modulating cellular lipid and lipoprotein metabolism (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4Ji Z.S. Brecht W.J. Miranda R.D. Hussain M.M. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1993; 268: 10160-10167Abstract Full Text PDF PubMed Google Scholar, 5Ji Z.S. Fazio S. Lee Y.L. Mahley R.W. J. Biol. Chem. 1994; 269: 2764-2772Abstract Full Text PDF PubMed Google Scholar, 6Ji Z.S. Mahley R.W. Arterioscler. Thromb. 1994; 14: 2025-2032Crossref PubMed Google Scholar, 7Stauderman M.L. Brown T.L. Balasubramaniam A. Harmony J.A.K. J. Lipid Res. 1993; 34: 190-200Abstract Full Text PDF PubMed Google Scholar). The function of apoE synthesized in multiple extrahepatic cell types remains incompletely defined. In macrophages, the regulation of apoE gene expression and apoE secretion by small increments in cellular cholesterol mass led to the hypothesis that apoE could serve a role in macrophage cholesterol homeostasis, as part of a regulatory loop to reduce cellular free cholesterol content (8Mazzone T. Gump H. Diller P. Getz G.S. J. Biol. Chem. 1987; 262: 11657-11662Abstract Full Text PDF PubMed Google Scholar). Subsequent studies confirmed such a role and indicated that expression of apoE in macrophages functions to enhance cholesterol efflux and reduce macrophage cholesterol stores in the presence and absence of extracellular acceptor particles (9Kruth H.S. Skarlatos S.I. Gaynor P.M. Gamble W. J. Biol. Chem. 1994; 269: 24511-24518Abstract Full Text PDF PubMed Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar). Studies using genetically engineered mice and bone marrow transplantation technology have confirmed an important role for macrophage-derived apoE in modulating susceptibility to atherosclerosis (11Linton M.F. Atkinson J.B. Fazio S. Science. 1995; 267: 1034-1037Crossref PubMed Scopus (411) Google Scholar, 12Boisvert W.A. Spangenberg J. Curtiss L.K. J. Clin. Invest. 1995; 96: 1118-1124Crossref PubMed Scopus (185) Google Scholar, 13Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar, 14Fazio S. Babaev V.R. Murray A.B. Hasty A.H. Carter K.J. Gleves L.A. Atkinson J.B. Linton M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar). Mice with a specific defect in macrophage apoE expression displayed a 10-fold increase in susceptibility to atherosclerosis while on a high fat diet (14Fazio S. Babaev V.R. Murray A.B. Hasty A.H. Carter K.J. Gleves L.A. Atkinson J.B. Linton M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar). Conversely, macrophage-specific expression of apoE has been shown to protect mice from the effect of atherogenic hyperlipidemia (13Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar). There are multiple properties of apoE which can be considered in evaluating the mechanism of such a protective effect (1Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 2Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). However, modulation of macrophage cholesterol content by endogenously synthesized apoE probably contributes to this important role in determining susceptibility of the vessel wall to atherogenic insult. In light of the apparent importance of macrophage-derived apoE in vessel wall homeostasis, it is imperative to fully understand the regulation of apoE expression in macrophages. Sterol modulation of apoE synthesis and secretion has been demonstrated in multiple macrophage types, including human monocyte-derived macrophages and mouse peritoneal macrophages (8Mazzone T. Gump H. Diller P. Getz G.S. J. Biol. Chem. 1987; 262: 11657-11662Abstract Full Text PDF PubMed Google Scholar, 15Mazzone T. Basheeruddin K. Poulos C. J. Lipid Res. 1989; 30: 1055-1064Abstract Full Text PDF PubMed Google Scholar, 16Wang-Iverson P. Gibson J.C. Brown W.V. Biochim. Biophys. Acta. 1985; 834: 256-262Crossref PubMed Scopus (19) Google Scholar, 17Kayden G.J. Maschio F. Traber M.G. Arch. Biochem. Biophys. 1985; 239: 388-395Crossref PubMed Scopus (33) Google Scholar, 18Cader A.A. Steinberg F.M. Mazzone T. Chait A. J. Lipid Res. 1997; 38: 981-991Abstract Full Text PDF PubMed Google Scholar). Increased apoE expression has been demonstrated after cholesterol loading of cells with modified lipoproteins or exposure of cells to cholesterol and oxysterol (8Mazzone T. Gump H. Diller P. Getz G.S. J. Biol. Chem. 1987; 262: 11657-11662Abstract Full Text PDF PubMed Google Scholar, 15Mazzone T. Basheeruddin K. Poulos C. J. Lipid Res. 1989; 30: 1055-1064Abstract Full Text PDF PubMed Google Scholar, 16Wang-Iverson P. Gibson J.C. Brown W.V. Biochim. Biophys. Acta. 1985; 834: 256-262Crossref PubMed Scopus (19) Google Scholar, 17Kayden G.J. Maschio F. Traber M.G. Arch. Biochem. Biophys. 1985; 239: 388-395Crossref PubMed Scopus (33) Google Scholar, 18Cader A.A. Steinberg F.M. Mazzone T. Chait A. J. Lipid Res. 1997; 38: 981-991Abstract Full Text PDF PubMed Google Scholar). Previous studies have indicated that sterol modulation of apoE synthesis and secretion is associated with a substantial increase in apoE gene transcription, with reports showing up to a 10-fold increase in apoE mRNA levels with a similar increase in apoE gene transcription rates (15Mazzone T. Basheeruddin K. Poulos C. J. Lipid Res. 1989; 30: 1055-1064Abstract Full Text PDF PubMed Google Scholar). In the current studies, we wished to address the issue of a potential post-transcriptional site for modulation of apoE secretion by sterols. A post-translational site was of particular interest based on previous observations that a significant portion of newly synthesized apoE in the macrophage is degraded prior to its release from the cell (19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar). To approach this issue, we reasoned that a post-transcriptional and post-translational effect would be more easily demonstrable in cells in which the large transcriptional response of the apoE gene to sterols could not obscure other potential regulatory loci. We therefore utilized a J774 cell macrophage model, which was transfected to constitutively express a human apoE cDNA. Expression of the apoE cDNA in this cell line is mediated by the human metallothionine IIa promoter and has been previously characterized in detail (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar, 20Lucas M. Iverius P.-H. Strickland D.K. Mazzone T. J. Biol. Chem. 1997; 272: 13000-13005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). These cells secrete 0.9–3.5 μg/ml/mg of cell protein of apoE over 24 h depending on culture conditions, an amount similar to that reported for human monocyte-derived macrophages. [35S]Methionine (10 Ci/mmol) and [32P]dCTP (800 Ci/mmol) were purchased from Amersham Corp. ALLN, 1The abbreviations used are: ALLN,N-acetyl-Leu-Leu-norleucinal; ALLM,N-acetyl-Leu-Leu-methioninal; ER, endoplasmic reticulum; Ac-LDL, acetylated low density lipoprotein. ALLM, and chloroquine were obtained from Sigma. Lactacystin was obtained from Biomol. 25-Hydroxycholesterol was obtained from Steraloids. All other materials were from sources described previously (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar, 20Lucas M. Iverius P.-H. Strickland D.K. Mazzone T. J. Biol. Chem. 1997; 272: 13000-13005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). J774 cells stably transfected to constitutively express a wild type human apoE cDNA have been characterized previously in detail (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar, 20Lucas M. Iverius P.-H. Strickland D.K. Mazzone T. J. Biol. Chem. 1997; 272: 13000-13005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Cells were selected and maintained in G418 (400 μg/dl) until 1 week prior to the initiation of experiments. Cells were grown in 10% fetal bovine serum and Dulbecco's modified Eagle's medium until the start of the described experimental incubations. Freshly isolated human monocytes were purified by elutriation. The elutriated cell population used for experiments was >95% monocytic, as determined by differential counts of Wright-stained smears. Human monocyte macrophages were plated and grown as described previously (21Hongwei D. Li Z. Mazzone T. J. Clin. Invest. 1995; 96: 915-922Crossref PubMed Scopus (52) Google Scholar). Cells were incubated with sterols dissolved in ethanol vehicle (final concentration < 0.5%). Control cultures were incubated in equivalent amounts of vehicle only. All steps were carried out at 4 °C. Cultures to be fractionated on continuous sucrose gradients were washed two times and scraped from the culture flasks with a rubber policeman in 5 mm sodium phosphate (pH 7.5), 0.1m sucrose and incubated on ice for 20 min to swell the cells. Cell suspensions were homogenized using 100 strokes in a Dounce homogenizer, and the sucrose concentration of the cell homogenate was adjusted to 20%. A linear sucrose gradient was prepared by mixing 6 ml of 20% sucrose (mixed with the cell homogenate) and 5.5 ml of 56% sucrose and centrifuging at 35,000 rpm for 18 h in a Beckman SW41 rotor, as described previously (22Lange Y. Muraski M.F. J. Biol. Chem. 1988; 263: 9366-9373Abstract Full Text PDF PubMed Google Scholar). Eleven fractions of equal volume were collected from the bottom of the tube, and the density of the fractions was monitored by comparing the refractive index of each fraction with a standard curve of density versus refractive index. Fractions were assayed immediately for marker enzyme activity. Recovery of enzyme activity and total labeled protein approximated 70% on these gradients. Isolation of intermediate density, nonlysosomal cell fractions was based on previously published methods (23Croze E.M. Morre D.J. J. Cell. Physiol. 1984; 119: 46-57Crossref PubMed Scopus (65) Google Scholar, 24Hamilton R.L. Moorehouse A. Havel E.J. J. Lipid Res. 1991; 32: 529-543Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Cultures to be fractionated were washed two times and scraped from the culture flasks with a rubber policeman in homogenizing medium (37.5 mm Tris-HCl, 0.5 m sucrose, 1% dextran, 5 mm MgC12, 0.1 mmphenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, pH 6.5). Cells in suspension were homogenized using 100 strokes in a Dounce homogenizer. The homogenate was then centrifuged at 2,000 rpm in a Beckman SW41 rotor for 5 min, followed by 10,000 rpm for 30 min. The upper one-half of the pellet was resuspended in 2 ml of homogenizing medium and then layered onto 9 ml of ice-cold 1.2 m sucrose and centrifuged at 25,000 rpm for another 30 min in a SW41 rotor. Two closely spaced opaque bands at the interface were recovered with a Pasteur pipette and diluted 20-fold with 1 × phosphate-buffered saline. Lysosomes were removed by centrifuging at 5,000 rpm for 20 min in a SW41 rotor and recovering the pellets. Pelleted membranes were assayed immediately for marker enzyme activity. Immunoprecipitations and Western and Northern blot hybridizations were performed as described previously in detail (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 15Mazzone T. Basheeruddin K. Poulos C. J. Lipid Res. 1989; 30: 1055-1064Abstract Full Text PDF PubMed Google Scholar, 19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar, 20Lucas M. Iverius P.-H. Strickland D.K. Mazzone T. J. Biol. Chem. 1997; 272: 13000-13005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 21Hongwei D. Li Z. Mazzone T. J. Clin. Invest. 1995; 96: 915-922Crossref PubMed Scopus (52) Google Scholar). ApoE was biosynthetically labeled with [35S]methonine (100–200 μCi/ml), and pulse-chase incubations were carried out as described previously. ApoE bands in SDS-polyacrylamide gel electrophoresis of immunoprecipitates were localized using a radiofluorescent image scanner and quantitated using ImageQuant software. Northern and Western blot signals were measured using a Molecular Dynamics laser densitometer with ImageQuant software. The method used to measure the degradation of total proteins or apoE in subcellular fractions was based on previously published methods (25Wang C.-N. Hobman T.C. Brindley D.N. J. Biol. Chem. 1995; 270: 24924-24931Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 26Barrett A.J. Kembhavi A.A. Brown M.A. Kirschke H. Knight C.G. Tamai M. Hanada K. Biochem. J. 1982; 201: 189-198Crossref PubMed Scopus (922) Google Scholar) with minor modifications. After isolation of fractions at 4 °C, equal aliquots were mixed with half volumes of 0.4 m sodium potassium phosphate (pH 6.8) containing 8 mm dithiothreitol. Equal portions of each fraction were then incubated for an additional 3 h at 40 °C or kept at 4 °C. At the end of that time, total labeled protein was measured by trichloroacetic acid precipitation, and apoE was measured by Western blot hybridization or immunoprecipitation. The difference between the amount of protein present in a fraction after incubation at 40versus 4 °C was taken as the amount degraded during the 3-h incubation period. ApoE in immunoprecipitates and Western blots was quantitated, as detailed above, using ImageQuant software. The amount of apoE present at the end of the 4 °C incubation was assigned a value of 100%, and the amount at the end of the 40 °C incubation was expressed as a fraction of the 4 °C value. In our experiments, the percentage of apoE degraded ranged from 68 to 73% in control cells and from 27 to 43% in sterol-treated cells. Protein, free cholesterol, cholesterol ester, and phospholipid were measured as described previously (3Lucas M. Mazzone T. J. Biol. Chem. 1996; 271: 13454-13460Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Mazzone T. Reardon C. J. Lipid Res. 1994; 35: 1345-1353Abstract Full Text PDF PubMed Google Scholar, 15Mazzone T. Basheeruddin K. Poulos C. J. Lipid Res. 1989; 30: 1055-1064Abstract Full Text PDF PubMed Google Scholar,19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar, 20Lucas M. Iverius P.-H. Strickland D.K. Mazzone T. J. Biol. Chem. 1997; 272: 13000-13005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 21Hongwei D. Li Z. Mazzone T. J. Clin. Invest. 1995; 96: 915-922Crossref PubMed Scopus (52) Google Scholar). Galactosyltransferase (Golgi marker), acid phosphatase (lysosomal marker), 5′-nucleotidase (plasma membrane marker), and cytochrome c reductase (ER marker) were assayed as described previously (22Lange Y. Muraski M.F. J. Biol. Chem. 1988; 263: 9366-9373Abstract Full Text PDF PubMed Google Scholar, 25Wang C.-N. Hobman T.C. Brindley D.N. J. Biol. Chem. 1995; 270: 24924-24931Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In this series of studies, we used a preincubation protocol that included sterol and oxysterol. We first wished to evaluate apoE mRNA levels in response to this experimental preincubation to confirm the absence of a transcriptional response and eliminate the possibility that post-transcriptional stabilization of apoE mRNA levels occurred in response to the sterol/oxysterol preincubation. The results of a Northern blot hybridization, shown in Fig. 1, indicate that 24-h preincubations in sterol/oxysterol produced no change in apoE mRNA levels. We next conducted a series of pulse-chase experiments after a 24-h preincubation in sterol/oxysterol. The results of a representative experiment are shown in Fig.2. Cells preincubated in sterol/oxysterol show a 1.7-fold increase and a 1.5-fold increase in medium apoE after 30 and 60 min of chase, respectively. Comparable increases were observed in multiple experiments conducted in a similar manner. Examination of the cellular content of apoE at 0, 30, and 60 min of chase showed no difference. These results indicate that the preincubation in sterol/oxysterol could enhance apoE secretion at a translational or post-translational locus. The apoE that was not secreted in control cells did not appear to be retained within the cell and, therefore, was probably degraded. Given previous observations regarding the significant rate of degradation of newly synthesized apoE (19Mazzone T. Pustelnikas L. Reardon C.A. J. Biol. Chem. 1992; 267: 1081-1087Abstract Full Text PDF PubMed Google Scholar), these results were consistent with the hypothesis that the sterol/oxysterol preincubation modified the degradation rate for newly synthesized apoE and thereby enhanced its secretion. We therefore directly investigated the effect of the sterol/oxysterol incubation on the degradation rate of apoE.Figure 2Post-transcriptional modulation of apoE secretion by sterols. Cells were plated and grown as described under “Experimental Procedures.” For the experiment, cells were incubated in growth medium alone or in the same medium with 1 μg/ml of 25-hydroxycholesterol (25-OH CH) and 10 μg/ml of cholesterol (CHOL) for 24 h prior to the start of a 30-min labeling period. Cultures were then chased for 30 or 60 min. Equal numbers of trichloroacetic acid-precipitable dpm were used for immunoprecipitation of apoE from cell culture media (upper panel) and cell lysates (lower panel) at 0, 30, and 60 min of chase time. ApoE was quantitated as described under “Experimental Procedures,” and the values shown are the mean ± S.D. from triplicate cultures.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addressing the potential for modification of apoE degradation by the sterol/oxysterol preincubation, we first wished to examine the subcellular distribution of apoE in control cells and cells preincubated in sterol/oxysterol and to determine which subcellular fractions were primarily responsible for degrading newly synthesized apoE. To study the subcellular distribution of newly synthesized apoE and the distribution of the activity for degrading it, macrophage cell homogenates were fractionated on 23–42% continuous sucrose gradients. On these gradients, the plasma membrane was contained in the three most buoyant fractions, the ER marker was found in the three densest fractions, and the lysosome and Golgi markers were found in five fractions of intermediate density and were incompletely resolved. A predominantly nonglycosylated isoform of apoE was recovered from the dense fractions containing ER, whereas larger glycosylated forms were easily detectable in Golgi/lysosome and plasma membrane fractions. Multiple experiments were performed utilizing cells harvested after a 30-min pulse label and no chase or after a 30-min pulse and 30-min chase period. We did not detect a consistently significant difference between the distribution of apoE in control cells and cells preincubated in sterol/oxysterol (not shown). We next assayed each of the fractions for its ability to degrade newly synthesized apoE. We found that 60–70% of total apoE degradation occurred in intermediate density fractions in both control and sterol/oxysterol-treated cells (not shown). We therefore undertook a more focused study of apoE degradation and the effect of sterols in an intermediate density cell fraction. An increasingly important role has been ascribed to nonlysosomal degradative pathways in the turnover of newly synthesized protein. We therefore undertook a set of studies to evaluate the role of a nonlysosomal intermediate density compartment in the macrophage for the degradation of newly synthesized apoE and evaluate whether apoE degradation in this compartment could be modulated by the sterol/oxysterol preincubation. This intermediate density fraction, isolated by previously published techniques (23Croze E.M. Morre D.J. J. Cell. Physiol. 1984; 119: 46-57Crossref PubMed Scopus (65) Google Scholar, 24Hamilton R.L. Moorehouse A. Havel E.J. J. Lipid Res. 1991; 32: 529-543Abstract Full Text PDF PubMed Google Scholar), had the characteristics shown in Table I. The fraction was enriched in the Golgi marker with an 8-fold increase in its specific activity and a 30% recovery. The specific activities for the ER, lysosome, and plasma membrane markers were essentially unchanged. Significantly, only 5.3% of the lysosomal marker activity was recovered in this fraction.Table ISpecific activity of marker enzymes in total cell homogenate and intermediate density nonlysosomal fractions prepared from macrophagesGolgiERLysosomePMProteinHomogenate Specific activity5.0 ± 0.81.5 ± 0.20.5 ± 0.17.3 ± 1.1Intermediate density fraction Specific activity42.2 ± 2.60.9 ± 0.10.7 ± 0.17.0 ± 0.9 Recovery (%)30.1 ± 5.72.7 ± 0.15.3 ± 1.08.7 ± 1.24.6 ± 0.8J774 macrophages were plated and grown as described in the legend to Fig. 2. An intermediate density nonlysosomal fraction was prepared, and marker enzyme activities were determined in this fraction and total cell homogenate as described under “Experimental Procedures.” Specific activities are given as nmol of [3H]galactose incorporated/h/mg of protein for UDP galactose:N-acetyl-glucosamine galactosyltransferase (Golgi marker enzyme); nmol of cytochrome c reduced/min/mg of protein for NADPH:cytochrome c reductase (ER marker enzyme); nmol ofp-nitrophenyl phosphate dephosphorylated/min/mg of protein for acid phosphatase (lysosome marker enzyme); and nmol of nucleotide hydrolyzed/min/mg of protein for 5′-nucleotidase (plasma membrane (PM) marker enzyme). Results are mean ± S.D. from six independent experiments. Open table in a new tab J774 macrophages were plated and grown as described in the legend to Fig. 2. An intermediate density nonlysosomal fraction was prepared, and marker enzyme activities were determined in this fraction and total cell homogenate as described under “Experimental Procedures.” Specific activities are given as nmol of [3H]galactose incorporated/h/mg of protein for UDP galactose:N-acetyl-glucosamine galactosyltransferase (Golgi marker enzyme); nmol of cytochrome c reduced/min/mg of protein for NADPH:cytochrome c reductase (ER marker enzyme); nmol ofp-nitrophenyl phosphate dephosphorylated/min/mg of protein for acid phosphatase (lysosome marker enzyme); and nmol of nucleotide hydrolyzed/min/mg of protein for 5′-nucleotidase (plasma membrane (PM) marker enzyme). Results are mean ± S.D. from six independent experiments. This intermediate density fraction degraded almost two-thirds of newly synthesized total cellular protein, suggesting the presence of a major cellular degradative pathway. Major cellular pathways for degrading proteins are believed to primarily involve lysosomes or proteasomes (27Jentsch S. Schienker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 28Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (785) Google Scholar, 29Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar). Our fractionation technique significantly excluded lysosomes; however, we wished to exc" @default.
• W2019796869 created "2016-06-24" @default.
• W2019796869 creator A5042498358 @default.
• W2019796869 creator A5047997095 @default.
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• W2019796869 date "1997-12-01" @default.
• W2019796869 modified "2023-09-27" @default.
• W2019796869 title "Degradation of Macrophage ApoE in a Nonlysosomal Compartment" @default.
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