Differential Mobilization of Newly Synthesized Cholesterol and Biosynthetic Sterol Precursors from Cells
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m212503200
ISSN1083-351X
AutoresSari Lusa, Sanna Heino, Elina Ikonen,
Tópico(s)Plant biochemistry and biosynthesis
ResumoPrevious work demonstrates that the biosynthetic precursor of cholesterol, desmosterol, is released from cells and that its efflux to high density lipoprotein or phosphatidylcholine vesicles is greater than that of newly synthesized cholesterol (Johnson, W. J., Fischer, R. T., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 25037–25046). Here we report that the release of individual precursor sterols varies with the efflux of newly synthesized zymosterol being greater than that of lathosterol and both exceeding that of newly synthesized cholesterol when using either methyl-β-cyclodextrin or complete serum as acceptors. The transfer of newly synthesized lathosterol to methyl-β-cyclodextrin was inhibited by actin polymerization but not by Golgi disassembly whereas that of newly synthesized cholesterol was inhibited by both conditions. Newly synthesized lathosterol associated with cellular detergent-resistant membranes more rapidly than newly synthesized cholesterol. Upon efflux to serum, newly synthesized cholesterol precursors associated with both high and low density lipoproteins. Stimulation of the formation of direct endoplasmic reticulum-plasma membrane contacts was accompanied by enhanced efflux of newly synthesized lathosterol but not of newly synthesized cholesterol to serum acceptors. The data indicate that the efflux of cholesterol precursors differs not only from that of cholesterol but also from each other, with the more polar zymosterol being more avidly effluxed. Moreover, the results suggest that the intracellular routing of cholesterol precursors differs from that of newly synthesized cholesterol and implicates a potential role for the actin cytoskeleton and endoplasmic reticulum-plasma membrane contacts in the efflux of lathosterol. Previous work demonstrates that the biosynthetic precursor of cholesterol, desmosterol, is released from cells and that its efflux to high density lipoprotein or phosphatidylcholine vesicles is greater than that of newly synthesized cholesterol (Johnson, W. J., Fischer, R. T., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 25037–25046). Here we report that the release of individual precursor sterols varies with the efflux of newly synthesized zymosterol being greater than that of lathosterol and both exceeding that of newly synthesized cholesterol when using either methyl-β-cyclodextrin or complete serum as acceptors. The transfer of newly synthesized lathosterol to methyl-β-cyclodextrin was inhibited by actin polymerization but not by Golgi disassembly whereas that of newly synthesized cholesterol was inhibited by both conditions. Newly synthesized lathosterol associated with cellular detergent-resistant membranes more rapidly than newly synthesized cholesterol. Upon efflux to serum, newly synthesized cholesterol precursors associated with both high and low density lipoproteins. Stimulation of the formation of direct endoplasmic reticulum-plasma membrane contacts was accompanied by enhanced efflux of newly synthesized lathosterol but not of newly synthesized cholesterol to serum acceptors. The data indicate that the efflux of cholesterol precursors differs not only from that of cholesterol but also from each other, with the more polar zymosterol being more avidly effluxed. Moreover, the results suggest that the intracellular routing of cholesterol precursors differs from that of newly synthesized cholesterol and implicates a potential role for the actin cytoskeleton and endoplasmic reticulum-plasma membrane contacts in the efflux of lathosterol. Virtually all the organs of the body synthesize cholesterol, with extrahepatic tissues accounting for a significant fraction of whole body sterol production (1Dietschy J.M. Turley S.D. Spady D.K. J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar). Cholesterol is synthesized from acetyl-CoA via the mevalonate pathway that initially produces farnesyl diphosphate, a precursor for squalene, dolichol, heme a, ubiquinone, and isoprenylated proteins. The committed step in cholesterol synthesis is the cyclization of squalene to lanosterol. From this compound, cholesterol is synthesized in a 19-step process involving the activity of nine different enzymes (2Risley J.M. J. Chem. Ed. 2002; 79: 377-384Crossref Scopus (37) Google Scholar). Recent data indicate that sterols regulate the pathway both at the early (i.e. via hydroxymethylglutaryl-coenzyme A reductase) and postlanosterol steps (3Kim J.H. Lee J.N. Paik Y.K. J. Biol. Chem. 2001; 276: 18153-18160Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The late steps of cholesterol synthesis can proceed via lathosterol and 7-dehydrocholesterol or via desmosterol to cholesterol. Interestingly, the relative importance of the two pathways may shift in vivo, e.g. during aging (4Lutjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. J. Lipid Res. 2002; 43: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Cholesterol biosynthesis is critically important for human development and cannot be compensated for by increasing the uptake of cholesterol from exogenous sources. This is exemplified by an increasing number of inborn errors of metabolism that are attributed to mutations in cholesterol biosynthetic enzymes (5Nwokoro N.A. Wassif C.A. Porter F.D. Mol. Genet. Metab. 2001; 74: 105-119Crossref PubMed Scopus (58) Google Scholar). The prototype of these disorders, Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency of 7-dehydrocholesterol reductase, the last step in cholesterol synthesis via the lathosterol pathway. More recently, other multiple malformation/mental retardation syndromes, including lathosterolosis, have been characterized (6Brunetti-Pierri N. Corso G. Rossi M. Ferrari P. Balli F. Rivasi F. Annunziata I. Ballabio A. Russo A.D. Andria G. Parenti G. Am. J. Hum. Genet. 2002; 71: 952-958Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In these patients, cholesterol precursors may constitute up to ∼10% of total cellular and plasma sterols. Importantly, cholesterol precursors are also found in normal human plasma, at concentrations roughly 1:1000 of that of cholesterol (7Bjorkhem I. Miettinen T. Reihner E. Ewerth S. Angelin B. Einarsson K. J. Lipid Res. 1987; 28: 1137-1143Abstract Full Text PDF PubMed Google Scholar, 8Vanhanen H.T. Blomqvist S. Ehnholm C. Hyvonen M. Jauhiainen M. Torstila I. Miettinen T.A. J. Lipid Res. 1993; 34: 1535-1544Abstract Full Text PDF PubMed Google Scholar). The plasma levels of lathosterol and desmosterol are commonly used as measures of the cholesterol biosynthetic activity of the individual. Recent data suggest that these values are highly heritable (9Berge K.E. von Bergmann K. Lutjohann D. Guerra R. Grundy S.M. Hobbs H.H. Cohen J.C. J. Lipid Res. 2002; 43: 486-494Abstract Full Text Full Text PDF PubMed Google Scholar, 10Gylling H. Miettinen T.A. J. Lipid Res. 2002; 43: 1472-1476Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and could potentially be used to predict individual responsiveness to the cholesterol-lowering regimen (11Miettinen T.A. Gylling H. Atherosclerosis. 2002; 164: 147-152Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 12Gylling H. Miettinen T.A. Atherosclerosis. 2002; 160: 477-481Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Cholesterol biosynthetic enzymes are localized in the cytosol as well as rough and smooth endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; BHK, baby hamster kidney; DRMs, detergent-resistant membranes; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; LDL, low density lipoprotein; LPDS, lipoprotein-deficient serum; TLC, thin layer chromatography; PBS, phosphate-buffered saline; MEM, minimal essential medium. both the rate-limiting and the last enzyme of the pathway (hydroxymethylglutaryl-CoA reductase and 7-dehydrocholesterol reductase, respectively) being integral membrane proteins of the ER (13Krisans S.K. Ann. N. Y. Acad. Sci. 1996; 804: 142-164Crossref PubMed Scopus (76) Google Scholar, 14Reinhart M.P. Billheimer J.T. Faust J.R. Gaylor J.L. J. Biol. Chem. 1987; 262: 9649-9655Abstract Full Text PDF PubMed Google Scholar, 15Moebius F.F. Fitzky B.U. Lee J.N. Paik Y.K. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1899-1902Crossref PubMed Scopus (191) Google Scholar). Several steps of the pathway also occur in peroxisomes. However, the absence of functional peroxisomes does not lead to deficiency of cholesterol biosynthetic enzymes (16Hogenboom S. Romeijn G.J. Houten S.M. Baes M. Wanders R.J. Waterham H.R. J. Lipid Res. 2002; 43: 90-98Abstract Full Text Full Text PDF PubMed Google Scholar). The transfer of cholesterol from its site of synthesis in the ER to the plasma membrane and extracellular acceptors has been investigated in a number of studies (for reviews see Refs. 17Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar and 18Maxfield F.R. Wustner D. J. Clin. Invest. 2002; 110: 891-898Crossref PubMed Scopus (281) Google Scholar). Instead, the transfer of sterol precursors has so far received little attention despite the pioneering observations by Lange et al. (19Lange Y. Echevarria F. Steck T.L. J. Biol. Chem. 1991; 266: 21439-21443Abstract Full Text PDF PubMed Google Scholar) and Johnson and co-workers (20Johnson W.J. Fischer R.T. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 25037-25046Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 21Phillips J.E. Rodrigueza W.V. Johnson W.J. J. Lipid Res. 1998; 39: 2459-2470Abstract Full Text Full Text PDF PubMed Google Scholar) that indicate clear differences in the behavior of cholesterol and its biosynthetic precursors. Lange and co-workers (22Lange Y. Muraski M.F. J. Biol. Chem. 1987; 262: 4433-4436Abstract Full Text PDF PubMed Google Scholar, 23Echevarria F. Norton R.A. Nes W.D. Lange Y. J. Biol. Chem. 1990; 265: 8484-8489Abstract Full Text PDF PubMed Google Scholar) reported that in fibroblasts at least three cholesterol precursors, lanosterol, zymosterol, and 7-dehydrocholesterol were highly concentrated in the plasma membrane. Moreover, newly synthesized zymosterol was found to move to the plasma membrane faster than cholesterol, with a half-time of 9 min (that of cholesterol being 18 min). In contrast, in McA-RH7777 cells the rate of transport of newly synthesized desmosterol was found to be roughly equal to that of cholesterol, with a half-time of ∼30 min for cholesterol and ∼40 min for desmosterol (24Baum C.L. Reschly E.J. Gayen A.K. Groh M.E. Schadick K. J. Biol. Chem. 1997; 272: 6490-6498Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Lange et al. (19Lange Y. Echevarria F. Steck T.L. J. Biol. Chem. 1991; 266: 21439-21443Abstract Full Text PDF PubMed Google Scholar) further reported that plasma membrane zymosterol turned over rapidly by internalization and became converted to cholesterol. On the other hand, Johnson et al. (20Johnson W.J. Fischer R.T. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 25037-25046Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) showed that sterol precursors were not only enriched in the plasma membrane but were rapidly effluxed from cells to high density lipoprotein and phosphatidylcholine vesicles. The major biosynthetic sterol released from Chinese hamster ovary cells was reported to be desmosterol or a closely related sterol. The rapid efflux of biosynthetic desmosterol was attributed to its more efficient desorption from the plasma membrane rather than to its more efficient delivery to the plasma membrane (21Phillips J.E. Rodrigueza W.V. Johnson W.J. J. Lipid Res. 1998; 39: 2459-2470Abstract Full Text Full Text PDF PubMed Google Scholar). In the present work, the synthesis, intracellular partitioning, and cellular release of cholesterol and its select precursors were further studied. The efflux of sterols to both methyl-β-cyclodextrin and to serum was analyzed. We report that the faster efflux compared with newly synthesized cholesterol is observed for lathosterol, the major sterol precursor in the circulation, but in particular for zymosterol. Moreover, we provide evidence suggesting that the faster efflux of lathosterol is coupled to a cellular circuit different from that of newly synthesized cholesterol and that its release to physiological acceptors can be modulated differently from that of newly synthesized cholesterol. Materials—Media and reagents for cell culture were from Invitrogen. Lipoprotein-deficient serum (LPDS) was prepared as in Ref. 43Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1287) Google Scholar. [4-14C]Cholesterol (specific activity, 55.0 mCi/mmol), [3H]acetic acid (specific activity, 10.0 Ci/mmol), Redivue Pro [35S]Met/Cys labeling mixture (specific activity, 1,000 Ci/mmol), protein A-Sepharose, and Amplify Fluorographic Reagent were from Amersham Biosciences. Brefeldin A (BFA) was from Epicentre Technologies and lovastatin from Merck Sharp & Dohme. Jasplakinolide was kindly provided by Prof. Phillip Crews (Dept. of Chemistry and Biochemistry, Univ. of California, Santa Cruz). Cycloheximide, protease inhibitors, blue dyed latex beads, mevalonic acid lactone (mevalonate), methyl-β-cyclodextrin, cholesterol, and other unlabeled lipids were from Sigma with the exception of zymosterol, which was from Steraloids. Petroleum ether was from Fischer Scientific; all other solvents (HPLC-grade) and silica gel 60 TLC plates were from Merck. Anti-human albumin was from DAKO. Cell Culture—Baby hamster kidney (BHK)-21 clone 13 cells (ATCC CRL8544) were cultured in Glasgow's modified Eagle's medium (GMEM), 10 mm Hepes pH 7.4, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10% tryptose phosphate broth, 10% fetal bovine serum (complete BHK-medium), HuH7 cells as in Ref. 25Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Cancer Res. 1982; 42: 3858-3863PubMed Google Scholar, and NIH3T3 cells as in Ref. 26Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar. Where indicated, cells were sterol-starved by maintaining in growth medium supplemented with 5% LPDS instead of complete serum for 48 h prior to [3H]acetate labeling. Analysis of Sterol Biosynthesis and Efflux to Methyl-β-cyclodextrin— Cells were grown on 55-mm dishes in LPDS-containing medium supplemented with [14C]cholesterol (20 nCi/ml) for 48 h. [14C]Cholesterol labeling provides an internal standard for controlling the extent of material losses and cyclodextrin extraction during the lipid analyses as described in Ref. 27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar. The cells were washed with phosphate-buffered saline (PBS) and labeled with [3H]acetate (250 μCi/ml) in MEM, 10 mm Hepes pH 7.4, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, (experiment medium) for 5 or 15 min at 37 °C, followed by chasing in experiment medium containing 10 μm lovastatin and 25 mm mevalonate at 37 °C. For cyclodextrin extractions, the cells were incubated with 5 mm methyl-β -cyclodextrin during the last 5 min of chase in MEM supplemented with 10 mm Hepes pH 7.4, 0.35 g/liter NaHCO3, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (air medium) on a shaking water bath at 37 °C. The medium was collected, and the cells were scraped into ice-cold PBS, harvested by centrifugation, and resuspended in 2% NaCl. Aliquots of the medium and the cell suspension were analyzed by liquid scintillation counting to determine the [14C]cholesterol content. The lipids from the cells and the medium were extracted and analyzed by thin layer chromatography (TLC), silver ion HPLC, and liquid scintillation counting as previously described (27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar). Where indicated, jasplakinolide was added to the experiment medium to a final concentration of 3 μm (from a 3 mm stock in methanol) for 1 h and BFA to a final concentration of 5 μg/ml (from a 5 mg/ml stock in ethanol) for 15 min before [3H]acetate labeling. The labeling, chase, and cyclodextrin extraction were performed in the continued presence of the drugs. Efflux of Cholesterol and Biosynthetic Sterol Intermediates to Serum—Cells were grown on 55-mm dishes in LPDS-containing medium supplemented with [14C]cholesterol (20 nCi/ml) for 48 h. The cells were then washed with PBS, labeled with [3H]acetate in experiment medium (250 μCi/ml) for 5 or 15 min at 37 °C and chased for 30 min to 4 h in experiment medium containing 20% human serum, 10 μm lovastatin, and 25 mm mevalonate at 37 °C. The medium and the cells were collected as above, and aliquots of both were analyzed by liquid scintillation counting to determine the [14C]cholesterol content. Lipids from the medium and cells were extracted and separated by TLC and HPLC. Notably, as various [3H]acetate-derived cellular products were released to serum, the medium was analyzed by TLC prior to HPLC. The procedural losses were corrected for based on the recovery of the [14C]cholesterol label as in Ref. 27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar. Where indicated, the serum-containing chase medium was supplemented with 0.8-μm diameter latex beads (1:10 dilution of a 10% bead suspension). Analysis of Albumin Secretion—Confluent HuH7 cultures in 25-mm diameter wells were preincubated with Met- and Cys-free MEM supplemented with 10 mm Hepes pH 7.4, 0.35 g/liter NaHCO3, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin for 1 h at 37 °C. The cells were then pulse-labeled for 10 min with [35S]Met/Cys labeling mixture (100 μCi/ml). Chase was performed in 1 ml of serum-free culture medium containing 10-fold excess of unlabeled Met and Cys and 20 μg/ml cycloheximide. Where indicated jasplakinolide (3 μm) was present during the preincubation, pulse and chase. The dishes were then placed on ice, and the medium was collected. The cells were washed with PBS, lysed in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, and 25 μg/ml each of chymostatin, leupeptin, antipain, and pepstatin, and insoluble material was removed by centrifugation. The medium and cell lysates were then incubated with anti-albumin antibodies for 16 h at 4 °C. The immunocomplexes were captured by protein A-Sepharose (2 h at 4 °C), and the bound material was washed five times with 10 mm Tris-HCl, pH 7.4, 0.1% SDS, 0.1% Triton X-100, and 2 mm EDTA. The proteins were boiled in reducing Laemmli sample buffer, resolved by SDS-PAGE (8% gels), and the albumin bands quantitated by Fujifilm BAS-1500 Imaging system. Triton X-100 Extraction—HuH7 cells were labeled with [14C]cholesterol for 48 h and with [3H]acetate for 5 min, and chased for 5–60 min as described above. The cells were washed and scraped in ice-cold PBS, harvested by centrifugation, resuspended in 1% Triton X-100-containing buffer on ice, and fractionated in 0–40% Optiprep flotation gradient in the presence of 1% Triton X-100 as described previously (27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar). Six fractions were collected from the top, and the lipids extracted and analyzed as above. Size-exclusion Chromatography—Serum lipoproteins were fractionated using Superose 6HR column (Amersham Biosciences) with PBS as elution buffer. The flow rate was 0.5 ml/min and 1-min fractions were collected. One-tenth of each fraction was used to determine the [3H]acetate-derived and [14C]cholesterol radioactivity. The major peaks of [3H]acetate-derived radioctivity were pooled, and lipids extracted and analyzed by TLC followed by HPLC as above to resolve the biosynthetic sterols. Procedural losses were corrected based on the recovery of [14C]cholesterol. Other Methods—Protein concentrations were measured according to Lowry (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Statistical significance of differences was determined using the Student's t test. Analysis of Cholesterol and Its Biosynthetic Precursors in Cell Lines—Newly synthesized cholesterol is often resolved only by TLC although this method is inadequate to separate cholesterol from its biosynthetic precursors as reported in several studies (20Johnson W.J. Fischer R.T. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 25037-25046Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 23Echevarria F. Norton R.A. Nes W.D. Lange Y. J. Biol. Chem. 1990; 265: 8484-8489Abstract Full Text PDF PubMed Google Scholar, 27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar, 29Burki E. Logel J. Sinensky M. J. Lipid Res. 1987; 28: 1199-1205Abstract Full Text PDF PubMed Google Scholar, 30Hokland B.M. Slotte J.P. Bierman E.L. Oram J.F. J. Biol. Chem. 1993; 268: 25343-25349Abstract Full Text PDF PubMed Google Scholar). To evaluate the resolving power of TLC, we pulse-labeled sterol-starved BHK cells with [3H]acetate for 15 min, chased for increasing times in the presence of lovastatin and excess unlabeled mevalonate (to stop further [3H]acetate incorporation into sterols), and analyzed the extracted lipids by TLC. The cholesterol TLC spot was then analyzed by silver ion HPLC and the fraction of [3H]cholesterol plotted. As shown in Fig. 1a, the fraction of [3H]cholesterol increased with increasing chase time but most of the [3H] radioactivity in the TLC cholesterol spot was actually not cholesterol. The result is in line with that reported by Johnson et al. (20Johnson W.J. Fischer R.T. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 25037-25046Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) using CHO cells and a longer [3H]acetate labeling time. Prolonged incubation at 14 °C during [3H]acetate labeling has been used to accumulate newly synthesized cholesterol intracellularly prior to chasing at 37 °C (26Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 31Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Therefore, the fraction of cholesterol present in the TLC spot was also monitored under these conditions. At chase times under 30 min when cholesterol was postulated to undergo rapid movement, less than 50% of the TLC spot represented [3H]cholesterol (Fig. 1b). According to HPLC analysis, [3H]lathosterol was one of the major [3H]acetate-derived products co-migrating in the TLC spot, representing 35% of the dpms analyzed at 0–10 min and 25% at 30 min of chase (data not shown). The result suggests that the use of TLC alone could yield misleading results and reinforces the necessity of using methods with high resolving ability for accurate separation of cellular sterols. Next, the levels of [3H]acetate-derived newly synthesized cholesterol and its biosynthetic precursor sterols were measured by HPLC from fibroblastic (BHK and NIH3T3) and hepatic (HuH7) cell lines. Prior to [3H]acetate labeling, the cells were sterol-starved by culturing for 2 days in lipoprotein-deficient medium. The cells were pulse-labeled with [3H]acetate for 15 min and chased for 30 or 60 min. Both the rate and efficiency of [3H]acetate incorporation into cholesterol varied considerably between similarly cultured cells (Fig. 2). HuH7 and NIH3T3 cells produced [3H]cholesterol more efficiently than BHK cells that were slower in synthesizing cholesterol and contained larger fractions of the precursor sterols. In BHK cells, [3H]zymosterol represented the major sterol peak by HPLC analysis both at 30 and 60 min of chase (Fig. 2, a and b). BHK cells also contained significant levels of both [3H]lathosterol and [3H]desmosterol, whereas in HuH7 and NIH3T3 cells, lathosterol represented the major precursor sterol, and only minor amounts of desmosterol were detected (Fig. 2b). The results in HuH7 cells are in line with those obtained in another hepatic cell line, HepG2, with cholesterol as the main biosynthetic sterol product (20Johnson W.J. Fischer R.T. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 25037-25046Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). On the other hand, some fibroblastic cells synthesize cholesterol efficiently while others do not, as exemplified by NIH3T3 and BHK cells, respectively. Efflux of Cholesterol and Its Biosynthetic Precursors to Methyl-β-cyclodextrin—To examine the efflux of newly synthesized sterols, the release of [3H]acetate-derived sterols to methyl-β-cyclodextrin was analyzed from BHK and HuH7 cells after increasing chase times. For HuH7 cells, the [3H]acetate labeling was shortened to 5 min to increase the proportion of radiolabeled precursor sterols. Methyl-β-cyclodextrin was added to the cells for 5 min at 37 °C either immediately after the labeling or after increasing chase times in serum-free medium (in the presence of statin and unlabeled mevalonate). The cyclodextrin concentration was titrated such that the efflux of prelabeled cellular [14C]cholesterol was ∼25%. The efflux of [3H]cholesterol increased gradually with increasing chase times in both cell lines, being somewhat faster in HuH7 than in BHK cells as shown previously (27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar) (Fig. 3a). In both cell lines, the efflux of newly synthesized [3H]lathosterol was greater than that of newly synthesized [3H]cholesterol at all time points analyzed (Fig. 3a). In BHK cells, the major precursor [3H]zymosterol was very efficiently recovered by cyclodextrin and was virtually absent from the cells, yielding a very high efflux percentage (82.4 ± 0.3% at 5 min, 80.5 ± 1.0% at 60 min, and 52.8 ± 2.39% at 120 min of chase). Accordingly, when the dpms of 3H-labeled sterols in the medium were plotted in BHK cells the overwhelming majority represented zymosterol at short chase times (5–60 min) (Fig. 3b). By contrast, [3H]desmosterol was least readily effluxed of the BHK cell sterol precursors, the [3H]desmosterol efflux percentage being intermediary between [3H]cholesterol and [3H]lathosterol (Fig. 3a). The more efficient completion of cholesterol biosynthesis in HuH7 cells was paralleled by the release of fewer precursors and correspondingly more of newly synthesized cholesterol to the acceptor (Fig. 3b). We have previously shown that the efflux of newly synthesized cholesterol to cyclodextrin is moderately inhibited in both BHK and HuH7 cells by BFA (27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar). However, we now observed that BFA had no effect on the cyclodextrin availability of newly synthesized lathosterol under the same conditions (Fig. 4, a and b). In search of additional modulators of newly synthesized sterol efflux we tested the effect of a membrane-permeant promoter of actin polymerization, the marine sponge toxin, jasplakinolide. We found that this compound inhibited slightly but reproducibly the efflux of both newly synthesized cholesterol and lathosterol (Fig. 4, a and b). Interestingly, for newly synthesized cholesterol the effect was apparently additive with that of BFA, suggesting that jasplakinolide affected a Golgi-bypass route of cholesterol transport (Fig. 4a). This was also in line with the observation that the jasplakinolide treatment had no effect on albumin secretion from the cells (Fig. 4c). The combination of BFA and jasplakinolide was not significantly more effective than jasplakinolide alone in inhibiting the efflux of lathosterol (Fig. 4b). Similar inhibition by jasplakinolide on the efflux of newly synthesized lathosterol to methyl-β-cyclodextrin was observed in BHK cells (data not shown). Association of Newly Synthesized Lathosterol with Detergent-resistant Membranes—Next, the association of newly synthesized lathosterol and cholesterol with detergent-resistant membrane fractions (DRMs) was compared. We have earlier shown that newly synthesized cholesterol was initially found in Triton X-100 soluble membranes but upon chasing, gradually associated with DRMs, kinetically closely paralleling its availability for efflux to cyclodextrin (27Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (210) Google Scholar). We now observed that newly synthesized lathosterol acquired detergent resistance more rapidly than newly synthesized cholesterol in the same cells, with 35–40% found in DRMs already at 5 min of chase while at that time point, only ∼20% of newly synthesized cholesterol was detergent resistant (Fig. 5). At 15 min of chase, the difference between the detergent resistance of newly synthesized lathosterol and cholesterol was still considerable (50–55% and 30–35% in DRMs, respectively; Fig. 5c) but started to level off at longer chase times (Fig. 5b). At 1–2
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