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• W2023202905 abstract "The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4–6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action. The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4–6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action. sterol regulatory element-binding protein Chinese hamster ovary fetal calf serum Dulbecco's modified Eagle's medium human lipoprotein-deficient serum phosphate-buffered saline sterol regulatory element-binding protein cleavage activating protein low density lipoprotein polyacrylamide gel electrophoresis The accumulation of cholesterol 1The trivial names used are: cholesterol, 5-cholesten-3β-ol; 3β-acetyl-25-hydroxycholesterol, 5-cholesten-3β,25-diol 3-acetate; 22(R)-hydroxycholesterol, 5-cholesten-3β,22(R)-diol; 25-hydroxycholesterol, 5-cholesten-3β,25-diol; 27-hydroxycholesterol, 5-cholesten-3β,27-diol; desmosterol, 5,24-cholestadien-3β-ol; 24(S),25-epoxycholesterol, 5-cholesten-24(S),25-epoxy-3β-ol; 25-fluorocholesterol, 5-cholesten-25-fluoro-3β-ol.1The trivial names used are: cholesterol, 5-cholesten-3β-ol; 3β-acetyl-25-hydroxycholesterol, 5-cholesten-3β,25-diol 3-acetate; 22(R)-hydroxycholesterol, 5-cholesten-3β,22(R)-diol; 25-hydroxycholesterol, 5-cholesten-3β,25-diol; 27-hydroxycholesterol, 5-cholesten-3β,27-diol; desmosterol, 5,24-cholestadien-3β-ol; 24(S),25-epoxycholesterol, 5-cholesten-24(S),25-epoxy-3β-ol; 25-fluorocholesterol, 5-cholesten-25-fluoro-3β-ol. in cells activates a feedback mechanism that delimits further buildup of this essential but toxic lipid (1Edwards P.A. Ericsson J. Annu. Rev. Biochem. 1999; 68: 157-185Crossref PubMed Scopus (388) Google Scholar). Suppression is mediated by decreasing the production of active sterol regulatory element-binding proteins (SREBPs),2 transcription factors that are required for the expression of genes in the cholesterol supply pathways (2Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2937) Google Scholar, 3Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1092) Google Scholar). In the presence of excess sterols, SREBP is maintained as an inactive precursor in the membranes of the endoplasmic reticulum and transcription from supply pathway genes is low. Sterol loss activates a processing pathway in which the SREBP precursor is transported from the endoplasmic reticulum to the Golgi apparatus and therein cleaved by proteases to release the amino-terminal half of the protein from the membrane-bound precursor. The cleaved protein migrates to the nucleus and activates the transcription of the low density lipoprotein (LDL) receptor gene and genes encoding enzymes in the cholesterol biosynthetic pathway, leading to increased cellular cholesterol.cDNAs and genes encoding proteins that participate in this complex mechanism of feedback regulation have been identified recently. These include DNAs encoding two closely related SREBPs (1 and 2) (4Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (781) Google Scholar, 5Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (498) Google Scholar), an SREBP cleavage activating protein (SCAP) (6Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), which functions to retain SREBPs in the endoplasmic reticulum in the presence of sterols and to escort them to the Golgi in the absence of sterols (7Nohturfft A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (191) Google Scholar, 8Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and the Site 1 and Site 2 proteases of this subcellular compartment (9Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.Z. Chang T.Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 10Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). All of these proteins are tightly associated with the membranes of the cell, presumably to allow close monitoring of and response to fluctuations in the levels of bilayer sterols.It has been known for many years that oxysterols are far more potent than cholesterol in exerting negative feedback regulation on the sterol biosynthetic and uptake pathways (11Kandutsch A.A. Chen H.W. J. Biol. Chem. 1974; 249: 6057-6061Abstract Full Text PDF PubMed Google Scholar, 12Brown M.S. Goldstein J.L. J. Biol. Chem. 1974; 249: 7306-7314Abstract Full Text PDF PubMed Google Scholar). When dissolved in ethanol and added to the medium of cultured cells at concentrations of less than 1 μm, the oxysterols 25-hydroxycholesterol or 27-hydroxycholesterol inhibit the proteolysis of SREBP precursors, which in turn suppresses the synthesis of cholesterol and the uptake of LDL cholesterol via the LDL receptor. In contrast, the addition of cholesterol in ethanol causes at best only modest suppression. The mechanisms by which oxysterols mediate their potency have not been defined, nor has it proven straightforward to determine whether oxysterols play a regulatory role in the whole animal. Although several enzymes that synthesize (13Russell D.W. Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (303) Google Scholar) and degrade (14Schwarz M. Lund E.G. Lathe R. Bjorkhem I. Russell D.W. J. Biol. Chem. 1997; 272: 23995-24001Crossref PubMed Scopus (143) Google Scholar, 15Li-Hawkins J. Lund E.G. Bronson A.D. Russell D.W. J. Biol. Chem. 2000; 275: 16543-16549Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) oxysterols have been characterized, their physiological significance in vivoremains unproven.In a pioneering series of studies, Lin et al. (16Lin H.-S. Rampersaud A.A. Archer R.A. Pawlak J.M. Beavers L.S. Schmidt R.J. Kauffman R.F. Bensch W.R. Bumol T.F. Apelgren L.D. Eacho P.J. Perry D.N. McClure D.B. Gadski R.A. J. Med. Chem. 1995; 38: 277-288Crossref PubMed Scopus (27) Google Scholar, 17Lin H.-S. Rampersaud A.A. Beavers L.S. McClure D.B. Gardner A.J. Eacho P.I. Foxworthy P.S. Gadski R.A. Steroids. 1999; 64: 735-741Crossref PubMed Scopus (3) Google Scholar) exploited the potency of oxysterols and knowledge of the feedback regulation of cholesterol metabolism to identify small molecules that modulate the system. To this end, libraries of compounds were screened using an assay that detected the reversal of oxysterol-mediated suppression of a human LDL receptor promoter reporter gene. Among the active compounds identified was the sterol LY295427 (Fig.1), which had an EC50 value in the low micromolar range. The closely related compound LY306309, differing only in the stereochemistry at carbon 3 (Fig. 1), was inactive. When administered to cholesterol-fed hamsters, LY295427 reduced plasma LDL levels by as much as 69% (18Bensch W.R. Gadski R.A. Bean J.S. Beavers L.S. Schmidt R.J. Perry D.N. Murphy A.T. McClure D.B. Eacho P.I. Breau A.P. Archer R.A. Kauffman R.F. J. Pharmacol. Exp. Ther. 1999; 289: 85-92PubMed Google Scholar). Experiments with cultured cells and liver extracts prepared from treated hamsters indicated that LY295427 increased the levels of unidentified oxysterol-binding proteins (19Bowling N. Matter W.F. Gadski R.A. McClure D.B. Schreyer T. Dawson P.A. Vlahos C.J. J. Lipid Res. 1996; 37: 2586-2598PubMed Google Scholar); however, the relationship between these findings and the decrease in serum cholesterol levels was not elucidated.In the current studies we show that LY295427 reverses the oxysterol-mediated suppression of SREBP processing in cultured cells. The consequences of this reversal are the activation of multiple SREBP-responsive genes, including the LDL receptor gene, and a concomitant increase in the metabolism of LDL, thus explaining the hypocholesterolemic effects of the compound. When considered with thein vivo activity of LY295427, these findings support a role for oxysterols in the regulation of cholesterol metabolism in the whole animal.RESULTSThe consequences of LY295427 treatment for cellular cholesterol metabolism were determined by treating diploid human fibroblasts with 30 μm compound for 48 h and then measuring the binding, uptake, and degradation of [125I]LDL (TableI). In two separate experiments, cells grown in lipoprotein-deficient medium, conditions that result in the induction of LDL receptor expression, metabolized microgram amounts of LDL. The quantity of LDL metabolized in this medium was not altered by treatment with LY295427. Cells grown in the presence of a mixture of sterols (5 μg/ml cholesterol and 0.4 μg/ml 25-hydroxycholesterol) metabolized far less LDL because of oxysterol-mediated suppression of LDL receptor expression. Incubation of the sterol-treated cells with LY295427, however, completely restored the metabolism of LDL to levels observed in cells grown without sterols (Table I). When LDL or FCS were present in the medium, the amount of [125I]LDL metabolized was reduced, again via sterol-mediated suppression of LDL receptor expression, but LY295427 did not reverse this suppression (Table I).Table IEffects of LY295427 on LDL metabolism in human fibroblastsTreatment[125I]LDLBindingInternalizationDegradationng/4 h/mg cell proteinExperiment A None1085723433 LY2954271217434054 Sterols562691320 Sterols + LY2954271036493257 LDL2281378 LDL + LY29542717137522Experiment B None14910215233 LY29542713412354941 Sterols43241786 Sterols + LY2954271518974428 FCS27212650 FCS + LY29542747336908Human diploid fibroblasts were plated on day 0 at a density of 2 × 104 cells/6-cm dish in DMEM containing 10% (v/v) FCS. On day 2 the medium was replaced with fresh DMEM containing 10% (v/v) FCS. On day 5, the medium was removed, the cells were washed once with PBS, and fresh DMEM medium containing 10% (v/v) HLPPS supplemented with 30 μm LY295427, 0.4 μg/ml 25-hydroxycholesterol plus 5 μg/ml cholesterol (Sterols), 10 μg/ml LDL, or 10% (v/v) FCS as indicated, were added to the dishes. On day 7, the medium was removed, the cells were washed once with PBS, and DMEM containing 2 mg/ml BSA, the supplements described above, and 10 μg/ml [125I]LDL were added as indicated. The binding, uptake, and degradation of [125I]LDL were measured after a 4-h incubation as described (21Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1277) Google Scholar). The numbers shown (means of triplicate determinations) represent high affinity values, which were calculated by subtracting the values obtained in the presence of excess unlabeled LDL (500 μg/ml) from those obtained in the absence of unlabeled LDL. Open table in a new tab Sterols repress the synthesis of mRNA transcribed from the LDL receptor gene in SV589 cells, a line of transformed human fibroblasts (29Yamamoto T. Davis C.G. Brown M.S. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (970) Google Scholar). To establish whether LY295427 could overcome this suppression, SV589 cells were cultured in the presence and absence of sterols and the compound, and levels of LDL receptor mRNA were assessed by blotting (Fig. 2). SV589 cells grown in lipoprotein-deficient medium contained substantial amounts of LDL receptor mRNA (lane 1). As expected, the presence of sterols in the medium decreased the level of LDL receptor mRNA (lane 2). The addition of LY295427 in the absence of sterols did not alter levels of LDL receptor mRNA in cells grown in lipoprotein-deficient medium (lane 3); however, the addition of the compound in the presence of sterols completely prevented the suppression of this mRNA (lane 4). Under none of the examined conditions did levels of the cyclophilin mRNA change in these cells (Fig. 3).Figure 2Induction of LDL receptor mRNA by LY295427 in immortalized human fibroblasts. SV589 cells were set up on day 0 as described under “Experimental Procedures.” On day 1, the cells were refed medium E supplemented as indicated with sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) and/or 20 μm LY295427. After a further 24-h incubation, the cells were harvested, and poly(A)+ mRNA was isolated. Aliquots of mRNA (5 μg) from cells treated with vehicle (lane 1), sterols (lane 2), LY295427 (lane 3), or sterols and LY295427 (lane 4) were separated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA probes encoding the LDL receptor (GenBankTM accession number NM000527) or rat cyclophilin (GenBankTM accession number M19533). After washing, the filter was exposed to x-ray film for 4.5 h.kb, kilobases; LDLR, LDL receptor.View Large Image Figure ViewerDownload (PPT)Figure 3Time course of gene expression in LY295427-treated human SV589 cells. Dishes of SV589 cells were set up on day 0 as described under “Experimental Procedures.” On day 1, the cells were refed medium E supplemented with sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol) and 20 μm LY295427. The cells were harvested at time 0 (lane 1) and following 6 h (lane 2), 12 h (lane 3), or 24 h (lane 4) of incubation. Poly(A)+ mRNA was isolated, and 5-μg aliquots were analyzed by blot hybridization with 32P-labeled cDNA probes encoding the LDL receptor (GenBankTM accession number NM000527), SREBP-2 (GenBankTM accession number NM004599), mouse stearyl-CoA desaturase (GenBankTMaccession number BC007474), and rat cyclophilin (GenBankTMaccession number M19533). After washing, filters were exposed to x-ray film for 15–24 h. kb, kilobases; LDLR, LDL receptor.View Large Image Figure ViewerDownload (PPT)The ability of LY295427 to alter the expression of other sterol responsive genes was assessed in SV589 cells by cDNA microarray technology. RNA was prepared from cells grown in medium with sterols ± LY295427 and then converted into cDNA in the presence of a deoxynucleoside triphosphate derivatized with a fluorescent dye. Different dyes were used to label the minus and plus compound cDNA probes, which were subsequently hybridized to two different microarrays containing 8,000–10,000 human cDNAs. Changes in gene expression were measured by fluorescence spectroscopy, and the data were organized according to rank order differences in the levels of a given mRNA. As summarized in TableII, the 15 genes whose expression changed the most under these conditions were all involved in sterol or fatty acid metabolism. For example, the expression of the gene encoding stearoyl-CoA desaturase was induced 6–7-fold in SV589 cells grown in the presence of sterols plus LY295427 versus levels found in cells cultured in sterols minus LY295427. Other mRNAs, such as that encoding farnesyl diphosphate synthetase, were elevated by growth in the presence of LY295427 but to a lesser extent (1.8 to 2.1-fold; TableII).Table IIIdentification of mRNAs with increased expression after LY295427 treatment of human SV589 cellsGeneFunctionFold increaseGenBank™ accession numberExperiment 1Experiment 2Stearoyl-CoA desaturaseFatty acid biosynthesis7.36.5AB032261Methyl sterol oxidaseCholesterol biosynthesis6.9NDAA157955Isopentenyl diphosphate isomeraseCholesterol biosynthesis6.6NDH08899Farnesyl-diphosphate farnesyltransferaseCholesterol biosynthesis5.43.1X69141Mevalonate kinaseCholesterol biosynthesis4.91.5X753113-Hydroxy-3-methylglutaryl-CoA reductaseCholesterol biosynthesis4.53.0H16650Squalene epoxidaseCholesterol biosynthesis3.72.6AF098865LDL receptorCholesterol supplyND3.0NM_0005277-Dehydro-cholesterol ▵7-reductaseCholesterol biosynthesisND3.0BE378962Mevalonate diphosphate decarboxylaseCholesterol biosynthesis2.82.3U49260ATP citrate lyaseLipid biosynthesis2.31.4AW967351SREBP-2Transcription of lipid metabolism genesND1.9AA608556Isopentenyl-diphosphate δ-isomeraseCholesterol biosynthesisND1.9X17025Farnesyl diphosphate synthaseCholesterol biosynthesis2.11.8D14697Sterol C4-methyl oxidaseCholesterol biosynthesisND1.7NM_006745In Experiment 1, poly(A)+ mRNA isolated from SV589 cells grown in medium E supplemented with 1 μg/ml 25-hydroxycholesterol ± 20 μm LY295427 was subjected to hybridization analyses using a human microarray representing 10,000 cDNAs (Incyte Genomics, St. Louis, MO) as described under “Experimental Procedures.” In Experiment 2, the same SV589 poly(A)+ mRNA was subjected to hybridization analysis except that a custom cDNA chip arrayed at Tularik, Inc was used. ND, not detected, either because the cDNA for this gene product was not present on the microarray or because the hybridization signal for the cDNA was not above background. Open table in a new tab These results were confirmed and extended in a series of RNA blotting experiments carried out in SV589 cells grown in the presence of sterols and LY295427 for different lengths of time. As shown by the data in Fig. 3, mRNA levels for the LDL receptor, SREBP-2, and stearoyl-CoA desaturase, three genes identified in the cDNA array experiments, were low in cells grown in the presence of sterols. The amounts of these mRNAs began to increase 6 h after the addition of LY295427 to the sterol-containing medium, and they reached maximal levels at 24 h. In contrast, the levels of an mRNA transcribed from the cyclophilin gene, which is not sterol-responsive, remained unchanged throughout the time course of the experiment (Fig. 3).A majority of the genes whose expression was altered by LY295427 were targets of the SREBP-1 and SREBP-2 transcription factors (30Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (268) Google Scholar), suggesting that the compound in some manner affected the activity of these DNA-binding proteins. To test this hypothesis, we monitored the processing of SREBPs in CHO-7 cells treated with LY295427. When grown in the absence of sterols, CHO-7 cells contained ample amounts of both SREBP-1 and -2 precursor and mature forms (Fig.4, lanes 1 and 5). Conversely, when grown in the presence of sterols, CHO-7 cells contained the membrane-bound precursor but little or no nuclear SREBP (lanes 2 and 6). The addition of LY295427 did not affect levels of the SREBP precursors or their processed nuclear forms in cells grown in the absence of sterols (lanes 3 and7), but it completely prevented the decrease in the nuclear forms normally observed in the presence of sterols (lanes 4and 8).Figure 4LY296427 reverses sterol-mediated suppression of SREBP processing in CHO-7 cells. On day 0, the cells were set up as described under “Experimental Procedures.” On day 2, cells were refed medium C supplemented with vehicle (lanes 1 and5), sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol, lanes 2 and 6), 20 μmLY295427 (lanes 3 and 7), or LY295427 and sterols (lanes 4 and 8). The cells were harvested after 24 h, and fractions enriched in membrane and nuclear proteins were prepared by centrifugation. Aliquots of protein (40–65 μg) from each fraction were separated by SDS-PAGE, transferred to a nylon membrane, and incubated with 5 μg/ml rabbit anti-hamster SREBP-1 IgG-179 (lanes 1–4) or 5 μg/ml mouse anti-hamster SREBP-2 IgG-7D4 (lanes 5–8). In the left lower panel, the cleaved form of SREBP-1 in the nuclear fraction is indicated by anarrow, and an intense cross-reacting protein is indicated by an asterisk. The positions to which prestained protein markers migrated to in the polyacrylamide gel are indicated on the right of the lumigrams. The blots were developed with a chemiluminescence reagent and exposed to film for 5–15 s.View Large Image Figure ViewerDownload (PPT)LY295427 was similarly able to reverse the suppression of SREBP processing caused by 25-hydroxycholesterol in human SV589 cells. The data of Fig. 5 showed that as the concentration of this oxysterol was increased from 0 to 5 μg/ml there was a progressive decrease in the amount of SREBP-2 in the nuclear fraction. This decrease was prevented by inclusion of 20 μm LY295427 in the culture medium. The addition of LDL to the medium at concentrations ranging from 5 to 100 μg/ml also suppressed the processing of SREBP-2; however, LY295427 did not reverse this suppression (Fig. 5). The levels of the LDL receptor and HMG-CoA synthase mRNAs were monitored in this same experiment, and these correlated directly with the amounts of SREBP-2 present in each condition (data not shown). These results agreed with those presented in Table I and indicated that LY295427 reversed the inhibition of SREBP processing mediated by oxysterols but not by LDL.Figure 5LY295427 reverses oxysterol- but not LDL-mediated suppression of SREBP processing. Human SV589 cells were plated on day 0 of the experiment as described under “Experimental Procedures.” On day 1, the cells were refed medium E supplemented with 25-hydroxycholesterol (0.2–5.0 μg/ml, upper panels), LDL (5–100 μg/ml, lower panels), and 20 μm LY295427 as indicated. On day 2, the cells were harvested, a fraction enriched in nuclear proteins was prepared by differential centrifugation, and aliquots containing 80 μg of protein were analyzed by immunoblotting with a mouse monoclonal antibody (IgG-1D2) that recognized SREBP-2. Immune complexes were detected subsequently by chemiluminescence.View Large Image Figure ViewerDownload (PPT)The hydrolysis of cellular sphingomyelin by the enzyme sphingomyelinase triggers the movement of cholesterol from the plasma membrane to the endoplasmic reticulum, which in turn causes suppression of SREBP processing (27Scheek S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11179-11183Crossref PubMed Scopus (99) Google Scholar). To determine whether LY295427 could reverse this suppression, CHO-7 cells were treated with different concentrations of sphingomyelinase in the presence or absence of 20 μmcompound and SREBP-2 processing was followed in cell lysates (Fig.6). Suppression was observed when the level of sphingomyelinase exceeded 100 milliunits/ml (lanes 1–4), and the inclusion of LY295427 in the culture medium had no effect on this regulation (lanes 5–8).Figure 6LY295427 does not reverse suppression of SREBP processing caused by sphingomyelinase. CHO-7 cells were plated on day 0 as described under “Experimental Procedures.” The medium was removed on day 2, the cells were washed twice with PBS, and fresh medium supplemented with 10 μg/ml of an acylcholesterol acyl transferase inhibitor (Sandoz 58–035) and 20 μm LY295427 (as indicated) was added. After 30 min, sphingomyelinase was added to the medium at the indicated concentrations, and the incubation continued for 90 min. The cells were harvested, lysed with a buffer containing SDS, and aliquots (50 μg) of the resulting lysates were subjected to SDS-PAGE followed by immunoblotting. Precursor (P) and mature (M) forms of SREBP-2 were detected with a mouse anti-hamster SREBP-2 monoclonal antibody (IgG-7D4) and chemiluminescent reagents. mu, milliunits.View Large Image Figure ViewerDownload (PPT)Sterol-regulated proteolysis of SREBP involves the cooperative actions of the sterol-sensing protein SCAP and two cleavage enzymes, the Site 1 and Site 2 proteases (31Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar). To determine whether the ability of LY295427 to facilitate SREBP processing in the presence of sterols also required these proteins, the effect of the compound in CHO-7 cell lines lacking SCAP or the Site 1 protease was assessed (Fig.7). In control experiments, wild type CHO-7 cells grown in the presence of sterols contained only the SREBP-2 precursor (lane 1). The processing of this form to the active nuclear protein was induced when LY295427 was added to the sterol-containing medium (lane 2). In a line of CHO-7 cells deficient in SCAP (SRD13A cells), no SREBP-2 proteins or peptides were detected, regardless of the presence or absence of sterols or LY295427 (lanes 3–6). The absence of SCAP chaperone activity renders SREBP unstable in SRD13A cells (23Rawson R.B. DeBose-Boyd R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 28549-28556Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and thus these results suggested that LY295427 was not acting by increasing the stability of the transcription factor. CHO-7 cells deficient in the Site 1 protease (SRD12B cells) contained the precursor form of SREBP-2 when cultured in the presence of sterols (lane 8), and, as expected, these cells were unable to cleave the precursor into the active form when sterols were removed from the medium (lane 7). LY295427 was unable to induce SREBP cleavage when these cells were grown in the presence or absence of sterols (lanes 9 and 10). In no cell line was the level of a control protein (immunoglobulin heavy chain-binding protein) changed (Fig. 7, lower panel). Similar results were obtained when the processing of SREBP-1 was followed (data not shown). Together these data indicated that the cleavage of SREBP in the presence of sterols and LY295427 involved two of the same components that process the protein in the absence of sterols.Figure 7LY295427 action requires an intact SREBP processing system. On day 0, CHO-7, SRD-12B, or SRD-13A cells were set up as described under “Experimental Procedures.” On day 2, the cells were refed medium C supplemented with vehicle (ethanol), sterols (1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol), or sterols plus 20 μm LY295427, as indicated, for 24 h. The cells were harvested and lysed with a buffer containing SDS, and aliquots (80 μg) of the resulting lysates were subjected to SDS-PAGE followed by immunoblotting. Precursor (P) and mature (M) forms of SREBP-2 were detected with a mouse anti-hamster SREBP-2 monoclonal antibody (IgG-7D4) and a chemiluminescent system. Signals were removed from the blot, and the filter was subjected to a second round of immunoblotting using a rabbit anti-rat immunoglobulin heavy chain-binding protein (BIP) antibody to confirm that equal amounts of protein were analyzed in each lane (lower panel). Lumigrams were generated by exposing the treated filters to x-ray film for 5–15 s.View Large Image Figure ViewerDownload (PPT)Dose-response experiments indicated that micromolar concentrations of LY295427 were needed to reverse the suppressive effects of oxysterols on SREBP processing in SV589 cells (Fig.8). An increase in the nuclear form of SREBP-2 was first detected at 5 μm LY295427 (lane 4). Enhanced production of processed SREBP-2 correlated with the increase of LY295427 up to 60 μm (lanes 5–8), which represented the solubility limit of the compound. In marked contrast, 60 μm LY306039, the 3β-stereoisomer of LY295427, did not affect SREBP-2 processing (Fig. 8, lane 9).Figure 8Dose-response experiment to determine effective concentration of LY2954" @default.
• W2023202905 created "2016-06-24" @default.
• W2023202905 creator A5026186239 @default.
• W2023202905 creator A5039217535 @default.
• W2023202905 creator A5041941258 @default.
• W2023202905 date "2001-11-01" @default.
• W2023202905 modified "2023-10-18" @default.
• W2023202905 title "The Hypocholesterolemic Agent LY295427 Reverses Suppression of Sterol Regulatory Element-binding Protein Processing Mediated by Oxysterols" @default.
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