In nonhepatic cells, cholesterol 7α-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis
2000; Elsevier BV; Volume: 41; Issue: 8 Linguagem: Inglês
10.1016/s0022-2275(20)33443-x
ISSN1539-7262
AutoresGary M. Spitsen, Svein Dueland, Skaidrite K. Krisans, Casey J. Slattery, Jon H. Miyake, Roger J. Davis,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoCHO cells expressing the liver-specific gene product cholesterol-7α-hydroxylase showed a 6-fold increase in the biosynthesis of [14C]cholesterol from [14C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7α-hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased ~3-fold. Cells expressing cholesterol-7α-hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7α-hydroxycholesterol. While cells expressing cholesterol-7α-hydroxylase displayed increased SREBP-dependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7α-hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25-hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis. —Spitsen, G. M., S. Dueland, S. K. Krisans, C. J. Slattery, J. H. Miyake, and R. A. Davis. In nonhepatic cells, cholesterol-7α-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis. J. Lipid Res. 2000. 41: 1347–1355. CHO cells expressing the liver-specific gene product cholesterol-7α-hydroxylase showed a 6-fold increase in the biosynthesis of [14C]cholesterol from [14C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7α-hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased ~3-fold. Cells expressing cholesterol-7α-hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7α-hydroxycholesterol. While cells expressing cholesterol-7α-hydroxylase displayed increased SREBP-dependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7α-hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25-hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis. —Spitsen, G. M., S. Dueland, S. K. Krisans, C. J. Slattery, J. H. Miyake, and R. A. Davis. In nonhepatic cells, cholesterol-7α-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis. J. Lipid Res. 2000. 41: 1347–1355. The unique ability of the liver to synthesize bile acids from cholesterol provides the major quantitative pathway through which cholesterol is removed from the body of mammals via the excretion of biliary bile acid–phospholipid–cholesterol micelles (1Edwards P.A. Davis R.A. Isoprenoids, sterols and bile acids.in: Vance D.E. Vance J. New Comprehensive Biochemistry. 31. Elsevier, Amsterdam1996: 341-362Google Scholar). Bile acid synthesis transforms the hydrophobic cholesterol molecule into an amphipathic molecule capable of forming mixed micelles with many lipid-insoluble substrates. This is achieved by adding hydroxyl groups, reducing the double bond, and oxidatively cleaving the side chain of cholesterol to form a carboxylic acid that usually forms an amide with either taurine or glycine (1Edwards P.A. Davis R.A. Isoprenoids, sterols and bile acids.in: Vance D.E. Vance J. New Comprehensive Biochemistry. 31. Elsevier, Amsterdam1996: 341-362Google Scholar). The initial step controlling bile acid synthesis is catalyzed by the liver-specific gene product cholesterol 7α-hydroxylase (C7αH: EC 1.14.13.17), a cytochrome P-450 enzyme located in the endoplasmic reticulum (2Noshiro M. Okuda K. Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alphahydroxylase.FEBS Lett. 1990; 268: 137-140Google Scholar, 3Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. Cloning and regulation of cholesterol 7-alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis.J. Biol. Chem. 1990; 265: 8190-8197Google Scholar, 4Li Y.C. Wang D.P. Chiang J.Y.L. Regulation of cholesterol 7 alpha-hydroxylase in the liver: cloning, sequencing and regulation of cholesterol 7 alpha-hydroxylase mRNA.J. Biol. Chem. 1990; 265: 12012-12019Google Scholar). Bile acids are also produced by an alternative pathway involving the 7α-hydroxylation of oxysterols (5Schwarz M. Lund E.G. Russell D.W. 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To gain an understanding of the role of C7αH in regulating cellular cholesterol homeostasis, we have examined how the stable expression of C7αH altered the phenotype of Chinese hamster ovary (CHO) cells, which normally do not express this liver-specific gene product (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). Unexpectedly, in CHO cells, C7αH expression led to increased expression of the LDL receptor in spite of increases in the cellular content of both free and esterified cholesterol (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). Thus, as a result of C7αH expression the ability of cellular sterols to signal negative feedback regulation of the LDL receptor was attenuated (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). In the present study we examined how the expression of C7αH in CHO cells influences the expression of genes controlling cellular cholesterol homeostasis. Specifically, we examined how C7αH expression affected the processing of SREBP, the expression of several SREBP-regulated genes, the transcription of SREBP-dependent reporters, and the expression of ABC1. Our results show that cellular and exogenous sterols display distinct differences in their ability to influence expression of several genes contributing to regulation of cellular cholesterol homeostasis. All tissue culture supplies, chemicals, and radioactive chemicals were obtained from suppliers, as described (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). Cells were maintained in modified Eagle's medium with 1.5% fetal bovine serum and 3.5% newborn calf serum (MEM Plus serum, Gemini Bioproducts, Woodland, CA) at 37°C in an atmosphere of 5% CO2. The plasmid expressing rat C7αH (pCMV-7α) was generously supplied by D. Russell and was constructed with the expression vector pCMV2 and the cDNA for rat C7αH (3Jelinek D.F. Andersson S. Slaughter C.A. Russell D.W. Cloning and regulation of cholesterol 7-alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis.J. Biol. Chem. 1990; 265: 8190-8197Google Scholar). JD15 cells, expressing this plasmid and cotransfected with the neomycin resistance plasmid pRSVneo, were obtained and cultured as described (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). K1-7α cells were obtained by transfecting CHO-K1 cells with an expression plasmid (pcDNA3-7α), which contained the coding sequence of rat C7αH ligated into pcDNA3 (Invitrogen, San Diego, CA), as described (19Wang S-L. Du E. Martin T.D. Davis R.A. Coordinate regulation of lipogenesis and the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1.J. Biol. Chem. 1997; 272: 19351-19364Google Scholar). CHO-K1 cells transfected with pcDNA3-7α were screened for resistance to G418 (400 μg/mL) and then single cell cloned. Cells stably expressing an SREBP-activated HMG-CoA reductase promoter/luciferase reporter [pREDluc, generously supplied by T. Osborne (20Osborne T.F. Bennett M. Rhee K. Red 25, a protein that binds specifically to the sterol regulatory region in the promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase.J. Biol. Chem. 1992; 267: 18973-18982Google Scholar)], were obtained by transfecting CHO-K1 with pREDluc and pRSV-neo and by transfecting JD15 cells with pREDluc and a plasmid expressing hygromycin B phosphotransferase (21Blochlinger K. Diggelmann H. Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.Cell. Mol. Biol. 1984; 4: 2929-2931Google Scholar). The resulting cell lines (DH1, without C7αH and DH2, with C7αH), expressing pREDluc, were maintained in serum containing MEM with G418 (400 μg/mL) and hygromycin B (500 μg/mL). Before use in experiments, G418 and hygromycin B were removed prior to plating and growth (to 80% confluence). Cells were incubated in serum-containing culture medium with [2-14C]acetate (5 μCi; specific activity, 47 mCi/mol) for 2 h, extracted with chloroform–methanol, and separated on silica gel thin-layer chromatography (TLC) plates, and the radioactivity in sterols and sterol esters was quantitated as described (22Davis R.A. Hyde P.M. Kuan J.C. Malone M.M. Archambault S.J. Bile acid secretion by cultured rat hepatocytes. Regulation by cholesterol availability.J. Biol. Chem. 1983; 258: 3661-3667Google Scholar). One day prior to reaching 80% confluence, the cell medium was changed as described in the figure legends. Delipidated serum was obtained by the cabosil extraction procedure (23Borensztajn J. Getz G.S. Kotlar T.J. Uptake of chylomicron remnants by the liver: further evidence for the modulating role of phospholipids.J. Lipid Res. 1988; 29: 1087-1096Google Scholar). After culturing (as described in the figure legends), cell extracts were assayed for HMG-CoA reductase (24Sinensky M. Torget R. Edwards P.A. Radioimmune precipitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Chinese hamster fibroblasts. Effect of 25-hydroxycholesterol.J. Biol. Chem. 1981; 256: 11774-11779Google Scholar) and squalene synthase (25Shechter I. Klinger E. Rucker M.L. Engstrom R.G. Spirito J.A. Islam M.A. Boettcher B.R. Weinstein D.B. Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.J. Biol. Chem. 1992; 267: 8628-8635Google Scholar). Each assay was performed in duplicate from three individual culture dishes of cells. Protein concentrations were determined by dye-binding assay (Bio-Rad, Hercules, CA). Values are reported as the mean ± SD for three individual culture dishes. For the cholesterol C7αH activity assay microsomes were isolated and assayed by the method described in Straka et al. (26Straka M.S. Junker L.H. Zaccaro L. Zogg D.I. Dueland S. Everson G.T. Davis R.A. Substrate stimulation of 7alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.J. Biol. Chem. 1990; 265: 7145-7149Google Scholar). To estimate the relative copy numbers of the reporter plasmid, pRedluc, in the CHO cell lines stably transfected with it, genomic DNA was isolated from both DH1 and DH2 cell lines. Genomic DNA was isolated with a QIAamp kit (Qiagen, Chatsworth, CA). Approximately 107 cells were scraped from a 150-mm dish and washed with phosphate-buffered saline (PBS) and then resuspended in 0.2 mL of PBS. To this, proteinase K and 0.2 mL of Qiagen solution AL were added and the solution was mixed by vortexing. The lysate was incubated at 70°C for 10 min and then applied to the QIAamp column and subjected to centrifugation in a microcentrifuge at 8,000 rpm for 1 min. The column was washed twice with Qiagen buffer AW according to the manufacturer instructions. The genomic DNA was eluted with 10 mm Tris-HCl, pH 9.0, by centrifugation in a microcentrifuge at 8,000 rpm for 1 min. The optical absorbance of the DNA was read at 260 and 280 nm and found to have an OD260/OD280 ratio of greater than 1.90. Genomic DNA (5.0 μg) from DH1 and DH2 cells was subjected to digestion with restriction endonucleases BamHI and Bgl II and analyzed for luciferase plasmid relative to endogenous C7αH by Southern blotting (27Leighton J.K. Dueland S. Straka M.S. Trawick J. Davis R.A. Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.Mol. Cell. Biol. 1991; 11: 2049-2056Google Scholar). After washing, the image was read and quantitated on a Molecular Dynamics (Sunnyvale, CA) PhosphorImager. To control for equal loading of samples, the gel was stained with ethidium bromide prior to blotting. After hybridization with the luciferase probe, the blot was stripped and reprobed with a probe recognizing the endogenous C7αH gene. Quantitation demonstrated that the molar ratio of DH1 to DH2 DNA was 0.86, in accordance with both the original optical density measurements and with staining of the loaded DNA. Transfections were performed by lipofection under optimized conditions. Typically, CHO-K1 cells were transfected with 200 ng of the farnesyl diphosphate synthase (FPPS) promoter-luciferase reporter construct, a kind gift from P. Edwards (28Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.Proc. Natl. Acad. Sci. USA. 1996; 93: 945-950Google Scholar), in the presence of 1 μL of LipofectAMINE (Life Technologies, Gaithersburg, MD) per well in a 12-well plate according to the manufacturer instructions. Transfection efficiencies were normalized by cotransfecting with pRL-TK (Promega, Madison, WI), a control vector containing a sea pansy (Renilla reniformis) luciferase gene driven by a thymidine kinase (TK) promoter in the molar ratio of 1:10. After incubation of cells as described in the figure legends, the medium was removed, and the cells were washed twice with 1× PBS. The cells were harvested by scraping with a rubber policeman in 1× lysis buffer (enhanced luciferase kit; Analytical Luminescence Laboratory, San Diego, CA). The lysate was centrifuged at 5,000 rpm for 5 min at 4°C to pellet the cellular debris, and the supernatant was transferred to a new tube. Luciferase activity of each sample was measured in triplicate, using an Analytical Luminescence Laboratory Monolight 2010 luminometer and reagents supplied by the manufacturer. Protein concentrations were determined by the method of Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). The production of light was linear with respect to time and protein throughout the assay. For the dual luciferase assay, after incubation of cells as described in the figure legends, the medium was removed, and the cells were washed twice with 1× PBS. The cells were lysed with 1× lysis buffer and the luciferase activity of each sample was measured in triplicate with an Analytical Luminescence Laboratory Monolight 2010 luminometer and reagents supplied by Promega. Cells were washed twice with PBS. PBS (1 mL) was added to each plate and then the cells were scraped into tubes. A portion of the cells was assayed for protein by the method of Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). To another portion, β-sitosterol was added as an internal standard and the cells were extracted with chloroform – methanol 1:1 (v/v) and stored under N2 until use. The solvent was evaporated under a stream of N2 and the samples were then resuspended in isopropanol. Samples were injected into a Hewlett-Packard (Palo Alto, CA) gas chromatograph and cholesterol mass was quantitated as described (30Davis R.A. Highsmith W.E. McNeal M.M. Schexnayder J.A. Kuan J.C. Bile acid synthesis by cultured hepatocytes. Inhibition by mevinolin, but not by bile acids.J. Biol. Chem. 1983; 258: 4079-4082Google Scholar). Poly(A)+ RNA was isolated from cells by a modification of the guanidinium isothiocyanate method (31Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thioguanate–phenol–chloroform extraction.Anal. Biochem. 1987; 162: 156-159Google Scholar), as described (32Trawick J.D. Lewis K.D. Dueland S. Moore G.L. Simon F.R. Davis R.A. Expression of cholesterol 7 alpha-hydroxylase by differentiated rat hepatoma l35 cells: inability to distinguish bile acid repression from cytotoxicity.J. Lipid Res. 1996; 37: 24169-24176Google Scholar). Two to 5 μg of the resulting mRNA was separated by 0.8% agarose-formaldehyde gel electrophoresis, transferred to a nitrocellulose membrane, UV cross-linked to nitrocellulose, and probed with nick-translated [32P]cDNA probes prepared from gel-purified inserts (19Wang S-L. Du E. Martin T.D. Davis R.A. Coordinate regulation of lipogenesis and the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1.J. Biol. Chem. 1997; 272: 19351-19364Google Scholar) and washed with a stringent SSC buffer (1× SSC is 0.15 m NaCl plus 0.015 m sodium citrate). Cells were harvested on ice in cold PBS containing a protease inhibitor cocktail (1 mm phenylmethylsulfonyl fluoride [PMSF], aprotinin [100 μg/mL], and leupeptin [50 μg/mL]), using a rubber policeman. Nuclei and membrane fractions were obtained as described (33Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. SREBP-1, a membrane-bound transcription factor released by a sterol-regulated proteolysis.Cell. 1994; 77: 53-62Google Scholar). Cells were centrifuged at 1,000 rpm for 10 min, and the pellets were resuspended in 10 volumes of cell homogenization buffer (10 mm HEPES-KOH at pH 7.6, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 1 mm EDTA, and the protease inhibitors described above). The cells were disrupted by passage through a 22-gauge needle (15 times) and then centrifuged at 1,000 rpm for 10 min at 4°C. The crude nuclear pellet was extracted with an equal volume of nuclear extraction buffer [20 mm HEPES-KOH at pH 7.6, 25% (v/v) glycerol, 0.5 m NaCl, 1.5 mm MgCl2, 1 mm EDTA, and the protease inhibitor cocktail] and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was used as nuclear extract for immunoblotting analysis. The microsomal membrane fraction was obtained by further centrifugation of the supernatant obtained from the nuclear pellet by ultracentrifugation at 45,000 rpm for 2 h at 4°C, using a TLA45 rotor (Beckman, Fullerton, CA). Western blotting was performed as described (19Wang S-L. Du E. Martin T.D. Davis R.A. Coordinate regulation of lipogenesis and the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1.J. Biol. Chem. 1997; 272: 19351-19364Google Scholar). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 1–15% gradient), the gels were electroblotted onto nitrocellulose membranes. The nonspecific binding sites of the membranes were blocked with 10% defatted dried milk, followed by addition of the appropriate primary antibody. The relative amount of primary antibody bound to the proteins on the nitrocellulose membrane was detected with the species-specific horse-radish peroxidase-conjugated IgG. Blots were developed by chemoluminescence with an ECL detection kit (Amersham, Arlington Heights, IL). The antibodies used are described in the figure legends. All values are reported as means ± SD. Statistical differences were calculated by using Student's t-test, double tailed. Values of P < 0.05 were considered to be significant. To obtain CHO cells that would stably express C7αH, CHO-K1 cells were cotransfected with plasmids expressing rat C7αH (pCMV-7a) and one that confers neomycin resistance (pRSVneo) (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). Cells were selected for resistance to both G418 (400 μg/mL) and 25-hydroxycholesterol, as described in detail (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). A representative clone (designated as JD15 cells) expressed C7αH mRNA and enzyme activity that was comparable to that of rat liver (18Dueland S. Trawick J.D. Nenseter M.S. MacPhee A.A. Davis R.A. Expression of 7-alpha-hydroxylase in non-hepatic cells results in liver phenotypic resistance of the low density lipoprotein receptor to cholesterol repression.J. Biol. Chem. 1992; 267: 22695-22698Google Scholar). JD15 cells were resistant to cytotoxic killing by 25-hydroxycholesterol, whereas CHO-K1 cells were sensitive (Fig. 1). To examine whether the resistance of JD15 cells to 25-hydroxycholesterol required C7αH, they were screened for25-hydroxycholesterol resistance in the presence of the cytochrome P-450 inhibitor ketoconazole (27Leighton J.K. Dueland S. Straka M.S. Trawick J. Davis R.A. Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.Mol. Cell. Biol. 1991; 11: 2049-2056Google Scholar). Whereas, by itself, ketoconazole had no apparent effect on the growth of JD15 cells, when 25-hydroxycholesterol was included in the medium, JD15 cells were rapidly killed (Fig. 1). These data show that the resistance of JD15 cells to 25-hydroxycholesterol cytotoxicity can be blocked by the cytochrome P-450 inhibitor ketoconazole. We have reported that JD15 cells expressed higher levels of LDL receptor mRNA than wild-type CHO-K1 cells did (32Trawick J.D. Lewis K.D. Dueland S. Moore G.L. Simon F.R. Davis R.A. Expression of cholesterol 7 alpha-hydroxylase by differentiated rat hepatoma l35 cells: inability to distinguish bile acid repression from cytotoxicity.J. Lipid Res. 1996; 37: 24169-24176Google Scholar). Unexpectedly, JD15 cells contained more free and esterified cholesterol than CHO-K1 cells did (32Trawick J.D. Lewis K.D. Dueland S. Moore G.L. Simon F.R. Davis R.A. Expression of cholesterol 7 alpha-hydroxylase by differentiated rat hepatoma l35 cells: inability to distinguish bile acid repression from cytotoxicity.J. Lipid Res. 1996; 37: 24169-24176Google Scholar). To examine the possibility that the increased cholesterol levels in JD15 cells were derived from de novo synthesis, we determined the relative rate of [14C]cholesterol synthesis from [14C]acetate (Fig. 2). When cultured in serum-containing medium, the relative rates of 14C-labeled cholesterol and cholesteryl esters were 8-fold and 6-fold greater, respectively, compared with the rates exhibited by CHO-K1 cells (Fig. 2).Fig. 2.[14C]Cholesterol biosynthesis by CHO cells. Wild-type CHO-K1 cells and JD15 cells were plated and grown to 80% confluence. [2-14C]Acetate (3.3 μCi/mL) was added to the medium and cells were incubated for 2 h. Cells were then harvested and lipids separated by TLC and the radioactivity was quantitated by β-scintillation analysis. Open columns represent wild-type CHO-K1 cells; hatched columns represent JD15 cells. Values represent the mean ± SD of three separate cell extracts in each group of cells. There were significant increases in both [14C]cholesterol and cholesteryl esters in JD15 cells compared with CHO-K1 cells (P < 0.001).View Large Image Figure ViewerDownload (PPT) To examine how the activities of cholesterol biosynthetic enzymes contribute to the increase in cholesterol biosynthesis, we measured the activities of two rate-limiting enzymes that act at the branch points of the isoprenoid (HMG-CoA reductase) and the sterol (squalene synthase) biosynthetic pathways (1Edwards P.A. Davis R.A. Isoprenoids, sterols and bile acids.in: Vance D.E. Vance J. New Comprehensive Biochemistry. 31. Elsevier, Amsterdam1996: 341-362Google Scholar). The activities of both HMG-CoA reductase and squalene synthase were increased
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