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Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells

2000; Elsevier BV; Volume: 41; Issue: 12 Linguagem: Inglês

10.1016/s0022-2275(20)32356-7

1539-7262

Reijiro Arakawa, Sumiko Abe-Dohmae, Michiyo Asai, Jin‐ichi Ito, Shinji Yokoyama,

Cancer, Lipids, and Metabolism

High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL. We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.—Arakawa, R., S. Abe-Dohmae, M. Asai, J-i. Ito, and S. Yokoyama. Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells. J. Lipid Res. 2000. 41: 1952–1962. High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL. We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.—Arakawa, R., S. Abe-Dohmae, M. Asai, J-i. Ito, and S. Yokoyama. Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells. J. Lipid Res. 2000. 41: 1952–1962. Cholesterol is not metabolized in peripheral cells except for the limited catabolism by sterol 27-hydroxylase and steroidogenesis in certain specific organs. Most cellular cholesterol is therefore removed and transported to the liver for its conversion to bile acids. High density lipoprotein (HDL) is believed to play a central role in this pathway. Two major mechanisms are proposed for the initial step of this transport system (1Yokoyama S. Apolipoprotein-mediated cellular cholesterol efflux.Biochim. Biophys. Acta. 1998; 1392: 1-15Google Scholar); nonspecific diffusion of cholesterol out of the cells through the aqueous phase and assembly of HDL by apolipoprotein with cellular lipids. Mutations were identified in ATP-binding cassette transporter 1 (ABC1) in patients with genetic HDL deficiency (2Brools-Wilson A. Marcil M. Clee S.M. Zhang L-H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O.F. Loubser O. Ouelette B.F.F. Fichter K. Ashbourne-Excoffon K.J.D. Sensen C.W. Schere S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Genest Jr., J. Hayden M.R. 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To date, the exact role of ABC1 in this reaction has not yet been fully understood, and its function in transmembrane cholesterol transport is proposed on the basis of the analogy to the function of other ABC transporters (12Young S.G. Fielding C.J. The ABCs of cholesterol efflux.Nat. Genet. 1999; 22: 316-318Google Scholar). Since we reported HDL assembly by extracellular apolipoproteins with cellular lipids (13Hara H. Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol.J. Biol. Chem. 1991; 266: 3080-3086Google Scholar), it has been demonstrated that this reaction involves at least two independent components (1Yokoyama S. Apolipoprotein-mediated cellular cholesterol efflux.Biochim. Biophys. Acta. 1998; 1392: 1-15Google Scholar); one is a cellular interaction site for helical apolipoprotein to assemble HDL particles with cellular phospholipid (14Li Q. Czarnecka H. Yokoyama S. Involvement of cellular surface factor(s) in lipid-free apolipoprotein-mediated cellular cholesterol efflux.Biochim. Biophys. Acta. 1995; 1259: 227-234Google Scholar, 15Smith J.D. Miyata M. Ginsberg M. Grigaux C. Shmookler E. Plump A.S. Cyclic AMP induces apolipoprotein E binding activity and promotes cholesterol efflux from macrophage cell line to apolipoprotein acceptors.J. Biol. Chem. 1996; 271: 30647-30655Google Scholar, 16Tsujita M. Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol.Biochemistry. 1996; 35: 13011-13020Google Scholar, 17Mendez A.J. Uint L. Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus.J. Lipid Res. 1996; 37: 2510-2524Google Scholar) and the other is a signal-initiated specific intracellular cholesterol mobilization system for its incorporation into HDL (18Mendez A.J. Oram J.F. Bierman E.L. Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.J. Biol. Chem. 1991; 266: 10104-10111Google Scholar, 19Li Q. Yokoyama S. Independent regulation of cholesterol incorporation into free apolipoprotein-mediated cellular lipid efflux in rat smooth muscle cells.J. Biol. Chem. 1995; 270: 26216-26223Google Scholar, 20Li Q. Tsujita M. Yokoyama S. Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cholesterol efflux in macrophages.Biochemistry. 1997; 36: 12045-12052Google Scholar, 21Mendez A.J. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion.J. Lipid Res. 1997; 38: 1807-1821Google Scholar). In an attempt to identify the mechanism in this specific intracellular cholesterol trafficking pathway, we found that an undifferentiated human monocytic leukemia cell line, THP-1, interacts with extracellular apolipoprotein A-I (apoA-I) and generates HDL with cellular phospholipid but no cholesterol. However, after the cells are differentiated by phorbol ester to the stage known to express various macrophage-like functions (22Tajima S. Hayashi R. Tsuchiya S. Miyake Y. Yamamoto A. Cells of a human monocytic leukemia cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase.Biochem. Biophys. Res. Commun. 1985; 126: 526-531Google Scholar, 23Hara H. Tanishita H. Yokoyama S. Tajima S. Yamamoto A. Induction of acetylated low density lipoprotein receptor on the cells of human monocytic leukemia cell line (THP-1).Biochem. Biophys. Res. Commun. 1987; 146: 802-808Google Scholar, 24Menju M. Tajima S. Yamamoto A. Expression of the apolipoprotein E gene in a human macrophage-like cell line, THP-1.J. Biochem. 1989; 106: 505-510Google Scholar, 25Nishimura N. Harada-Shiba M. Tajima S. Sugano R. Yamamura T. Qiang Q.Z. Yamamoto A. Acquisition of secretion of transforming growth factor-beta 1 leads to autonomous suppression of scavenger receptor activity in a monocyte-macrophage cell line, THP-1.J. Biol. Chem. 1998; 273: 1562-1567Google Scholar), cholesterol-rich HDL is generated by the cell-apolipoprotein interaction, indicating the induction of a reaction to incorporate cholesterol into the HDL. In the present article, we characterize the underlying mechanism of this phenomenon. THP-1 cells were maintained in RPMI 1640 (Iwaki, Tokyo, Japan) containing 10% fetal bovine serum (FBS) (GIBCO-BRL, Gaithersburg, MD), or its lipoprotein-deficient serum fraction (d > 1.23 g/ml) (LPDS) if so mentioned, in a humidified atmosphere of 5% CO2 and 95% air. For differentiation of THP-1 monocytes into macrophages, the cells were cultured in a six-well plate at a density of 3.0 × 106 cells/well in the presence of 3.2 × 10-7 M phorbol 12-myristate 13-acetate (PMA) (Wako, Osaka, Japan) for 72 h (23Hara H. Tanishita H. Yokoyama S. Tajima S. Yamamoto A. Induction of acetylated low density lipoprotein receptor on the cells of human monocytic leukemia cell line (THP-1).Biochem. Biophys. Res. Commun. 1987; 146: 802-808Google Scholar) except for the experiment to determine the effect of the length of the PMA treatment. The culture medium was replaced with RPMI 1640 supplemented with 0.2% bovine serum albumin (BSA) and incubated for a further 48 h. The undifferentiated cells were cultured in RPMI 1640 and 0.2% BSA for 48 h and seeded in a six-well plate at a cell density of 3.0 × 106/well. The differentiated and undifferentiated cells were cultured in the presence of lipid-free human apoA-I, apoA-II, or phosphatidylcholine-triolein microemulsion with a diameter of 25 nm (26Tajima S. Yokoyama S. Yamamoto A. Effect of lipid particle size on association of apolipoproteins with lipid.J. Biol. Chem. 1983; 258: 10073-10082Google Scholar), in 0.2% BSA-RPMI 1640 for 24 h. The lipid in the medium and the cells was extracted with chloroform–methanol 2:1 (v/v) and hexane–isopropanol 3:2 (v/v), respectively, for enzymatic determination of cholesterol (Kyowa Medics, Tokyo, Japan) and choline phospholipid (Wako). To measure the release of phosphatidylcholine and sphingomyelin, the cells were pulsed with [3H]choline chloride (Amersham, Arlington Heights, IL) for the last 48 h before incubating with apoA-I (13Hara H. Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol.J. Biol. Chem. 1991; 266: 3080-3086Google Scholar). Radioactivity of choline phospholipid released was counted after the lipid in the medium was extracted and separated by thin-layer chromatography. The cells were dissolved in 0.1 N NaOH for protein determination by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The apoA-I (20 μl/ml)-conditioned medium of the 12 wells (12 ml) was centrifuged at 1.64 × 105 g for 24 h at 4°C in a Hitachi (Tokyo, Japan) CP80b ultracentrifuge and the bottom 3.4 ml was collected. This fraction was overlaid on top of sucrose solutions of densities 1.31 g/ml (2.5 ml) and 1.15 g/ml (2.5 ml) in an ultracentrifuge tube for a Hitachi RP55T rotor. After centrifugation at 1.64 × 105 g for 48 h at 4°C, the solution was collected from the bottom into nine fractions. The contents of cholesterol and phospholipid as well as the density of each fraction were measured. Incorporation of [14C]oleic acid (0.45 μCi/ml; Amersham) into cholesteryl ester in the cells was determined (27Francis G.A. Oram J.F. Heinecke J.W. Bierman E.L. Oxidative tyrosylation of HDL enhances the depletion of cellular cholesteryl esters by a mechanism independent of passive sterol desorption.Biochemistry. 1996; 35: 15188-15197Google Scholar). After incubation of the cells with apoA-I at 20 μg/ml, for 2 h, the cells were incubated with [14C]oleic acid for an additional 1 h. The incorporation of radioactivity into the cholesteryl ester fraction was measured after lipid extraction and separation by thin-layer chromatography (method A). The microsomal fraction was prepared from THP-1 cells to measure its acyl-CoA:cholesterol acyltransferase (ACAT) activity (28Suckling K.E. Stange E.F. Dietschy J.M. Dual modulation of hepatic and intestinal acyl-CoA:cholesterol acyltransferase activity by (de-)phosphorylation and substrate supply in vitro.FEBS Lett. 1983; 151: 111-116Google Scholar). The cells were suspended in phosphate-buffered physiological saline and centrifuged at 650 g for 5 min to collect the pellet, to which 5 mM Tris-HCl (pH 8.5) was added and vortexed. The sample was chilled in ice and centrifuged at 650 g for 5 min, and the supernatant was centrifuged at 10,000 g for 30 min. The supernatant was collected and centrifuged at 105,000 g for 1 h to collect the pellet as the microsomal fraction. This fraction as 100 μg of protein was mixed with 250 nmol of cholesterol as cholesterol/phosphatidylcholine liposomes, and incubated in 0.1 M potassium phosphate buffer (pH 7.4) with [14C]oleoyl-CoA (0.1 μCi/2 nmol) at 37°C for 5 min (method B). Alternatively, [14C]cholesterol (1 μCi/50 nmol) was dissolved in 192 μl of 0.25% Triton WR-1339 and preincubated with 200 μg of microsomal protein in 8 μl at 37°C for 30 min, and the reaction was initiated by adding 30 nmol/4 μl of oleoyl-CoA at 37°C for 5 min (method C). Radioactivity in cholesteryl ester was counted after the lipid was extracted by chloroform–methanol 2:1. To evaluate an intracellular free cholesterol pool available for the ACAT reaction, the intracellular cholesterol esterification rate was standardized by the microsomal ACAT activity. The undifferentiated cells were suspended in 10% FBS-RPMI 1640 at a density of 5.2 × 107 cells/ml in the presence of a 20 mM concentration of the sense (5′-GCCAGCATGTCTGGGGG CAAATAC-3′), antisense (5′-GTATTTGCCCCCAGACATGCTG GC-3′), and scrambled (5′-TCGCACTTGTAGTTGGCCCGCA CA-3′) DNA for human caveolin-1, synthesized by ESPEC Oligo (Tsukuba, Japan). Control suspension contained cells without the oligonucleotides. Electroporation was carried out in a GenePulser (Bio-Rad, Hercules, CA) at 250 V, 1,100 μF, and then the cells were treated with PMA for differentiation. The apoA-I-mediated lipid release was measured as described above. The cells were harvested in 0.02 M Tris-buffered saline (pH 7.5) and pelleted by centrifugation at 600 g for 5 min. The cytosol and membrane fractions were separated by sonication of the cells in 20 mM phosphate buffer (pH 7.4) and centrifugation at 1.46 × 105 g as the supernatant and pellet, respectively. For the preparation of the detergent-resisting membrane (DRM) fraction (29Brown D.A. Rose J.K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.Cell. 1992; 68: 533-544Google Scholar), the cells were resuspended in 150 μl of 0.05 M boric acid buffer containing 0.15 M NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine (pH 7.2), and 3 ml of 0.02 M boric acid buffer containing 0.3 mM ethylenediaminetetraacetic acid (pH 10.2) was added. The suspension was strongly agitated for 10 min, mixed with 600 μl of 0.5 M boric acid (pH 10.2), and centrifuged at 600 g for 5 min. The supernatant was centrifuged at 12,500 g for 30 min, and the precipitate was sonicated in 0.02 M Tris-buffered saline (pH 7.5) containing the protease inhibitors and layered on the 35% sucrose solution. After centrifugation at 24,000 g for 1 h, the plasma membrane fraction at the interface was collected and sonicated in the 1% Triton X-100 and 8% sucrose solution, containing the protease inhibitors and 20 mM Tricine (pH 7.6), mixed with an equal volume of 60% sucrose, and centrifuged at 3.67 × 105 g for 2 h. The supernatant was diluted three times with 50 mM Tris buffer (pH 8.0) and the DRM fraction was precipitated by centrifugation at 3.67 × 105 g for 2 h. The protein content of each fraction was determined by the BCA method. Protein samples were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk at 4°C overnight. After incubation with rabbit anti-caveolin-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA), the membrane was washed three times, 15 min each in 0.02 M Tris-buffered saline containing 0.05% Triton X-100 (pH 7.5), and incubated with anti-rabbit IgG conjugated with horseradish peroxidase (BioSource International, Camarillo, CA). The membrane was washed and caveolin-1 was visualized by a chemiluminescence method. Total RNA was extracted by the standard acid guanidinium thiocyanate-phenol-chloroform method. Briefly, the cells were lysed in the presence of phenol and guanidium and then RNA was recovered in the aqueous phase by adding chloroform and subsequent centrifugation. RNA was precipitated with isopropanol, and the pellet was washed with ethanol and dried. Total RNA content was determined by measuring the optical absorbance ratio at 260 to 280 nm after the sample was dissolved in diethylpyrocarbonate-treated water. First-strand cDNA was synthesized from the total RNA, 5 μg, and the cDNAs of caveolin-1 and ABC1 were amplified in a SuperScript preamplification system (GIBCO-BRL) and by polymerase chain reaction (PCR) for 26 cycles by using Taq polymerase (TaKaRa Shuzo, Osaka, Japan). Glyceraldehyde-3-phosphate dehydrogenase cDNA was also amplified as an intracellular standard. The cDNA was visualized by SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR) after 2% agarose gel electrophoresis. The quantitativeness of the assay was validated by the linearity with respect to the dose of total RNA and the number of the PCR cycles. Figure 1 shows the effect of the differentiation of THP-1 cells by PMA on the removal of cellular cholesterol by apoA-I. The apoA-I-mediated cholesterol removal was induced by the PMA treatment of the cells in a time-dependent manner up to 72 h. ApoA-I-mediated HDL assembly was therefore analyzed in THP-1 cells after 72 h of PMA treatment (Fig. 2). When apoA-I was added to the culture medium, choline phospholipid was released into the medium from the cells in a dose-dependent manner in both the undifferentiated and differentiated stages, with a moderate increase in the differentiated condition. Cholesterol release by apoA-I was hardly detectable from the undifferentiated cells, whereas a substantial amount of cholesterol was released by apoA-I from differentiated cells. Further analysis of the choline phospholipid in the medium demonstrated that the release of phosphatidylcholine was hardly influenced by differentiation, whereas sphingomyelin release was increased in the same manner as cholesterol release. The HDL particles that appeared in the medium during the reaction were analyzed by density gradient ultracentrifugation. The HDL generated from undifferentiated cells contained a small amount of cholesterol whereas the HDL from differentiated cells was rich in cholesterol. The increase in HDL cholesterol was more than 15-fold in terms of the ratio to phospholipid.Fig. 2.Release of lipids by apoA-I from THP-1 cells. (A and B) Release of choline-phospholipid (PL) and cholesterol (Ch) mass into 1 ml of medium from undifferentiated and differentiated THP-1 cells (575 ± 7 and 639 ± 19 μg of cell protein per well, respectively) in the presence of apoA-I. (C and D) Release of choline-labeled phosphatidylcholine (PC) and sphingomyelin (SM) from undifferentiated and differentiated THP-1 cells (534 ± 13 and 555 ± 7 μg/well). (E and F) Density gradient ultracentrifugation analysis of the medium of undifferentiated and differentiated THP-1 cells (total 7.7 and 8.1 mg of cell protein, respectively) after incubation with apoA-I. The data represent the average ± SE of three experimental points for (A–D). Error bars are hidden by symbols for some data points. The data in (E) and (F) are the results of the average of duplicate assays for a single data point. DPM, radioactivity as decay per minute.View Large Image Figure ViewerDownload (PPT) ApoA-I-mediated cholesterol removal was analyzed in relation to the levels of cellular cholesterol. Differentiated cells contained a slightly higher amount of free and esterified cholesterol than undifferentiated cells, and it seems an extracellular supply of cholesterol, as the LPDS-conditioned cells had less cholesterol than the FBS-conditioned cells (Fig. 3, bottom). Cellular esterified cholesterol was decreased by apoA-I in differentiated cells, whereas cellular cholesterol did not change in undifferentiated cells, reflecting the lack of its removal by apoA-I. The apoA-I-mediated cholesterol removal from differentiated cells increased when cell cholesterol increased (Fig. 3, top left). When cholesterol was further loaded into differentiated cells by incubating them with acetylated low density lipoprotein (LDL) to the extent that cellular esterified cholesterol was 17.3 ± 1.1 μg/mg cell protein, the apoA-I-mediated release was up to 10.6 ± 0.4 μg/mg cell protein (data not shown). Because phospholipid removal did not change by cell cholesterol level (Fig. 3, top right), the HDL generated by this reaction was enriched in cholesterol as the cellular cholesterol increased in the differentiated THP-1 cells. These data were consistent with the finding by Bielicki, McCall, and Forte (30Bielicki J.K. McCall M.R. Forte T.M. Apolipoprotein A-I promotes cholesterol release and apolipoprotein E recruitment from THP-1 macrophage-like foam cells.J. Lipid Res. 1999; 40: 85-92Google Scholar). Cholesterol release from undifferentiated cells was again shown to be extremely low, whereas phospholipid release was essentially the same as in differentiated cells (Fig. 3, top). Cholesterol loading of undifferentiated cells via LDL resulted only in a slight increase in cellular cholesteryl ester. The apoA-I-mediated cholesterol release hardly increased and the reciprocal decrease in cellular cholesterol ester by apoA-I did not reach a detectable level (Fig. 4).Fig. 4.The effect of cholesterol loading of undifferentiated cells on apoA-I-mediated cholesterol removal and cellular cholesterol. The cells were incubated with LDL (200 μg/ml medium) for 24 h, washed, and then incubated with apoA-I (20 μg/ml) for 24 h. Total cholesterol in the medium and total and free cholesterol in the cell were measured as described in text. The esterified cholesterol was calculated as the difference between total and free cholesterol. Cell protein was 419.9 ± 7.8 μg/well of the cholesterol-unloaded cells and 363.1 ± 14.1 μg/well of the cholesterol-loaded cells.View Large Image Figure ViewerDownload (PPT) A cellular cholesterol compartment accessible by ACAT was probed (Table 1). Intracellular incorporation of radiolabeled oleic acid into cholesteryl ester was measured by using whole cells (Table 1, column A). The enzyme activity of the microsomes was measured separately by two independent methods because it can be altered by differentiation (Table 1, columns B and C). The compartment indicator (A) was then standardized by the enzyme activity (B and C). The enzyme activity increased twice by the PMA treatment and the ACAT-available cholesterol compartment remained the same after the standardization (A/B and A/C). This compartment decreased with the release of cholesterol by apoA-I only in differentiated cells, whereas the undifferentiated cells did not respond to apoA-I, reflecting the lack of cholesterol incorporation into the HDL assembled.TABLE 1.Incorporation of 14C[oleic] acid into cholesteryl ester and ACAT activity of the microsomal fraction in undifferentiated and differentiated THP-1 cellsaThe cells were incubated with apoA-I (20 μg/ml) for 2 h at 37°C and then [14C]oleic acid was added to the medium for the incubation for an additional 1 h to measure its incorporation into cholesteryl ester (A) (dpm/mg cell protein). The ACAT activity in the cells was estimated with the microsomal fraction as the cholesterol esterification rate by measuring incorporation of [14C]oleyl CoA into cholesteryl ester (B) and by measuring esterification of [14C]cholesterol (C) (pmol/min/mg microsomal protein) according to the method described in text. The values represent means ± SE of the triplicated assay. The A values are standardized by the B and C values in order to estimate the intracellular cholesterol compartment available for the ACAT reaction (A/B and A/C).ABCA/BA/CUndifferentiatedApoA-I (−)3,675 ± 30134.7 ± 0.614.2 ± 1.5105.9 ± 8.6258.8 ± 21.1ApoA-I (+)3,505 ± 34——101.0 ± 1.0246.8 ± 2.4DifferentiatedApoA-I (−)9,273 ± 63689.5 ± 0.831.9 ± 0.8103.6 ± 7.1290.7 ± 19.9ApoA-I (+)7,088 ± 213——79.2 ± 2.4bP < 0.01 from other A/B values.222.1 ± 6.7cP < 0.01 from other A/C values.a The cells were incubated with apoA-I (20 μg/ml) for 2 h at 37°C and then [14C]oleic acid was added to the medium for the incubation for an additional 1 h to measure its incorporation into cholesteryl ester (A) (dpm/mg cell protein). The ACAT activity in the cells was estimated with the microsomal fraction as the cholesterol esterification rate by measuring incorporation of [14C]oleyl CoA into cholesteryl ester (B) and by measuring esterification of [14C]cholesterol (C) (pmol/min/mg microsomal protein) according to the method described in text. The values represent means ± SE of the triplicated assay. The A values are standardized by the B and C values in order to estimate the intracellular cholesterol compartment available for the ACAT reaction (A/B and A/C).b P < 0.01 from other A/B values.c P < 0.01 from other A/C values. Open table in a new tab Figure 5 shows cholesterol release by apoA-II and by lipid microemulsion. ApoA-II-mediated cholesterol release was also induced by cellular differentiation by PMA. In contrast, cholesterol release induced by the lipid microemulsion by nonspecific diffusion was not influenced by differentiation. The finding with apoA-II was consistent with the previous view that the reaction to generate HDL from cellular lipids is not highly specific to apoA-I, but is mediated by many other helical apolipoproteins (31Hara H. Hara H. Komaba A. Yokoyama S. α-Helix requirement for free apolipoproteins to generate HDL and to induce cellular lipid efflux.Lipids. 1992; 2