Genetic Distinction between Sterol-mediated Transcriptional and ...

Vol. 266, No. 14, Issue of May 15,pp. 9128-9134, 1991 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY

(9 1991 by The American Society for Biochemistry and Molecular Biology., Inc.

Genetic Distinction between Sterol-mediated Transcriptional and Posttranscriptional Control of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase* (Received for publication, January 4, 1991)

Paul A. DawsonS, James E. Metherallt, NealeD. Ridgwayll, Michael S . Brown, and Joseph L. Goldstein From the Departmentof Molecular Genetics, University of Texas Southwestern Medical Center,Dallas, Texas 75235

Sterols reduce the activity of 3-hydroxy-3-methyl- in Ref. 1). The enzyme produces mevalonate, the bulk of glutaryl-coenzyme A reductase (HMG-CoA reductase) which is used for cholesterol synthesis. Small amounts of transcriptionally by inhibiting the synthesisof reduc- mevalonate are also required for synthesis of several lowtase mRNA and posttranscriptionally by accelerating abundance isoprenoid compounds, including ubiquinone, doldegradation of the enzyme. We and others have de- ichol, and prenylated proteins. When cultured cells are described mutant lines of Chinese hamster fibroblasts prived of cholesterol, the amount of HMG-CoA reductase that are completely resistant tosterol-mediated repres- protein increases by severalfold, primarily as a result of insion of transcription of HMG-CoA reductase as well as creased transcription of the gene. This increase is reversed two other sterol-regulated genes, HMG-CoA synthase and thelow density lipoprotein (LDL) receptor. In the when the cells are resupplied with cholesterol contained in current studies, we show that one lineof sterol-resist- low density lipoprotein (LDL), which enters cells via the LDL receptor. Suppression also occurs when cells are incubated ant mutant cells (SRD-3 cells) retains the ability to slow the degradationof HMG-CoA reductase by7-fold with oxygenated sterols, such as25-hydroxycholestero1,added in response to treatment with compactin, an inhibitor to themedium in ethanol. The complexity of the regulation of HMG-CoA reductase of reductase that blocks sterol synthesis. The compacis unmasked when cells are incubated with compactin, a tin effect is reversed by exogenous sterols. Similar results were obtained with another mutant line of competitive inhibitor of HMG-CoA reductase (1-3). The resterol-resistantcells(SRD-2 cells) whose defective sultant depletion of mevalonate deprives the cells not only of transcriptional regulationis attributable to a different sterols, but also of the nonsterol end products. As a result gene than that in the SRD-3 cells, as determined by there is a marked increase in HMG-CoA reductase protein complementation analysis. Thesedata indicate that thewhich is attributable to enhanced gene transcription, to acgene products that are defective in the SRD-3 and celerated translation of the reductase mRNA, and to slowed SRD-2 cells are not required for the sterol-mediated degradation of the protein. The compactin-mediated inducregulation of degradation ofHMG-CoA reductase. tion of gene transcription is completely reversed by LDL or Thus, mammalian cells possess at least two genetically 25-hydroxycholesterol. However, the rateof translation of the distinct mechanisms, one transcriptional and the othermRNA remains elevated unless the cells are given mevalonate posttranscriptional, for sensing and responding to the together with sterols. The slowed protein degradation is acintracellular level of sterols. celerated by sterols, but only when the concentration of compactin is low enough to allow the cells to produce a trace amount of mevalonate. These studies have led to theconcept The membrane-bound enzyme 3-hydroxy-3-methylglu- of multivalent regulation of HMG CoA reductase (1, 4). taryl-coenzyme A reductase (HMG-CoA reductase),’ a rate- Sterols control the rate of gene transcription, whereas other determining enzyme in cholesterol synthesis, is subject to mevalonate-derived products controlthe rate of mRNA transfeedback control at multiple levels in animal cells (reviewed lation. Reductase degradation is believed to be controlled by a combination of a sterol and a nonsterol product whose * This work was supported by Research Grant HL20948 from the synthesis canbe blocked only by a very high concentration of National Institutesof Health andby research grants from the Lucille compactin (1). P. Markey Charitable Trust and the Perot Family Foundation. The The mRNAs for several other proteins in the mevalonate costs of publication of thisarticle weredefrayed in part by the payment of pagecharges. Thisarticlemustthereforebe hereby pathway are also regulated by sterols. These include HMGmarked “advertisement” in accordance with 18 U.S.C. Section 1734 CoA synthase, the enzyme preceding HMG-CoA reductase solely to indicate thisfact. (5), andprenyltransferase, the enzyme that catalyzes the $ Recipient of Postdoctoral Fellowship H L 07524 from the Napolymerization of isoprene units (6). In addition, sterols contional Institutes of Health. trol the rateof transcription of the gene for the LDL receptor § Recipient of Postdoctoral Fellowship CA08398 from the National (7). To date there isno evidence that nonsterol products Institutes of Health. control any of these other proteins, nor is there convincing 1 Recipient of a Postdoctoral Fellowship from the Medical Research Council of Canada. evidence for posttranscriptional control. ’ The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglu- Sterol-mediated regulation of transcription of HMG-CoA taryl-coenzyme A; LDL, low density lipoprotein; SRD cells, mutant synthase and the LDL receptor is mediated by a common cell lines having a sterol-resistant defective phenotype; SRE-1, sterol sequence of 8-10 base pairs in the 5”flanking regions of both regulatoryelement-1; SDS, sodium dodecyl sulfate; CHO, Chinese hamster ovary; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic genes that is designated the sterol regulatory element-1 (SRE1) (1, 8). A similar sequence is present in the 5”flanking acid.

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Posttranscriptional Regulation

in Sterol-resistant Cells

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(5) that had been coupled to tuberculin-purified peptide derivative prior to immunization (16). Rabbit anti-HMG-CoA reductase was raised against bacterially-expressed COOH-terminal60-kDa cytosolic domain of human HMG-CoA reductase (which was kindly provided by K. L. Luskey of this Department). Theanti-reductase monoclonal antibody (IgG-AS)has been described previously (18). Other materials were obtained from previously reported sources (13). Cultured Cells-All cells were grown in monolayer at 37 “C in an atmosphere of 5% COz and were maintained in medium A (a 1:l mixture of Ham’s F-12 medium and Dulbecco’smodifiedEagle’s minimum essential medium containing 100 units/ml penicillin, 100 pg/ml streptomycin sulfate, 2 mM glutamine, and 10% (v/v)newborn calf lipoprotein-deficient serum). The SRD-2 and SRD-3 cells (see below) were maintained in medium A containing 1 and 2 pg/ml 25hydroxycholesterol, respectively. Mutagenesis and Isolation of 25-Hydroxycholesterol-resistant Cells-SRD-2 cells, a mutant line of 25-hydroxycholesterol-resistant CHO-7 cells, were isolated in 1987 as described previously (13). SRD3 cells were isolated by J. L. Goldstein and M. S. Brown in 1975 (but not previously reported) from V-79 cells that had been adapted to grow in 10% (v/v) calf lipoprotein-deficient serum. On day 0, V-79 cells were plated into seven 250-ml flasks (2 X lo5 cells/flask) in medium B (Ham’s F-12 medium supplemented with 1%(w/v) glutamine and 10% (v/v) calf lipoprotein-deficient serum). On day 2 the medium was changed to medium Bcontaining 1.2 mg/ml ethyl methanesulfonate. After 2 h at 37 “C the cells were washed with phosphate-buffered saline and replated into 60 dishes (100-mm) in medium B a t a density of 3 X lo5 cells/dish. After an expression period of 2 days, the mutagenized cells were subjected to selection in medium B containing 1 pg/ml 25-hydroxycholesterol. After several weeks, three surviving colonies were isolated with cloning cylinders and subjected to further selection with 2-5 pg/ml 25-hydroxycholesterol. The most resistant of the three clones is designated SRD-3. Once healthy cultures were established, the SRD-3 cells were maintained in medium A containing 2 pg/ml25-hydroxycholesterol. Genetic Complementation Analysis-Cells were plated on day 0 at 5 X 10’ cells/lOO-mm dish. On day 1, the cells were transfected with either pSV3 Neo (0.5 pg) (19) or pSV3 Hyg (0.5 pg) (kindly provided by M. Nakanishi of this Department) that had been coprecipitated with salmon sperm DNA (9.5 pg) and calcium phosphate (13). On day 2, the cells were fed medium A supplemented with either 700 pg/ ml G418 or 300 pg/ml hygromycin B. Cells were refed medium of identical composition every 3rd day for 12-14 days. G418-resistant or hygromycin-resistant colonies (200-1000 individual clones) were pooled and expanded in mass culture. Painvise fusion of G418resistant and hygromycin-resistant cells was performed by plating 8 X lo5 cells of each type in single wells of six-well Linbro plates on day 0 in medium A. On day 1, the cells werewashedtwice with phosphate-buffered saline, incubated with 0.5mlof polyethylene glycol 1500 (Boehringer Mannheim) for 1 min at room temperature, washed three times with 2 ml of a 1:l mixture of Ham’s F-12 medium and Dulbecco’s modified Eagle’sminimum essential medium containing 10% (v/v) dimethyl sulfoxide, and refed with medium A. On day 2, the cells were harvested and plated into four 100-mm dishes in medium A containing 700 pg/ml G418 and 300 pg/ml hygromycin in either the absence (plates A and B) or presence (plates C and D) of 1 pg/ml 25-hydroxycholestero1. The cells were refed every 2nd day with medium of identical composition. On days 7-14, the cells were washed, fixed, and stained with crystal violet. The total number of hybrids formed was determined by counting the number of colonies on plates A and B. The number of 25-hydroxycholesterol-resistant hybrids formed was determined by counting the number of colonies on plates Cand D. Assays-HMG-CoA reductase activity was measured in detergentEXPERIMENTALPROCEDURES solubilized cell extracts as described (15) and is expressed as pmol of Materials-25-Hydroxycholesterol and cholesterol were obtained [“CIHMG-CoA converted to [“C]mevalonate/min per mg of deterfrom Steraloids, Inc. Mevalonolactone was purchased from Fluka gent-solubilized protein. HMG-CoA synthase was assayed as deChemical Co. and converted to the sodium salt as described (2). scribed with several modifications (20). Briefly, cells werewashed Hygromycin B and G418 (Geneticin) were purchased from Calbio- twice and harvested in phosphate-buffered saline. Cell pellets were chem Corp. and GIBCO, respectively. Chinese hamster lung cells (V- lysed in 10 mM Tris-HC1 (pH 7.3), 1 mM EDTA, and 0.3 M sucrose 79 cells) were obtained from the Institute for Medical Research, by repeated aspiration through a 25-gauge needle. The homogenate Camden, NJ. Compactin was kindly provided by Akira Endo (Tokyo was centrifuged at 14,000 X g for 15 min at 4 “C, and the supernatant Noko University, Tokyo, Japan). Newborn calf lipoprotein-deficient was dialyzed for 12 h against 20mM potassium phosphate (pH 7), 0.5 serum (d > 1.215 g/ml) was prepared as described (15). T ~ ~ I I [ ~ ’ S ]mM dithiothreitol, and 0.1 mM EDTA. Dialyzed cytosols (30 pgof label (1100 Ci/mmol) was obtained from ICN Radiochemicals. [“C] protein) were assayed in a reaction mixture containing 0.1 M TrisAcetyl-coA (53 mCi/mmol) was obtained from DuPont-New England HC1,20 mM MgCl, 10 p M acetoacetyl-CoA, and 0.6 mM [l-’4C]acetylNuclear. Rabbit anti-HMG-CoA synthase was raised against the CoA (12,000 cpm/nmol) in afinal volume of 0.1 ml. The assay mixture COOH-terminal 15 amino acids of the hamster HMG-CoA synthase minus labeled substrate was preincubated for 2 min at 30 “C, and the

region of the HMG-CoA reductase gene, but the evidence for its role in sterol-mediated feedback regulation is not conclusive (1, 9). In the LDL receptor and HMG CoA synthase genes, SRE-1 appears to act asa conditional positive element. It enhances transcription in the absence of sterols and loses this effect when sterols are present (1, 9, 10). The putative protein that interacts with the SRE-1 has not yet been identified. Insight into transcriptional regulation of the mevalonate pathway has come from the study of mutagenized Chinese hamster fibroblasts that have lost sensitivity to sterol regulation (11-13). These cells are selected in culture by growth in theabsence of LDL and in thepresence of 25-hydroxycholesterol. Under these conditions the parentalcells die because 25-hydroxycholestero1 suppresses cholesterol synthesis but cannot replace cholesterol in cell membranes. Cells that have lost feedback control of the mevalonate pathway grow under these conditions because they continue to synthesize cholesterol. Although the 25-hydroxycholesterol-resistantcells are selected only for a failure to suppress cholesterol synthesis, they also fail to suppress the LDL receptor mRNA, suggesting that these cells are defective in a step that is common to regulated transcription of all sterol-suppressible genes (13). Moreover, spontaneousrevertants simultaneously regain their sensitivity to sterol-mediated repression of both HMGCoA synthase andreductase (14). Two lines of sterol-resistant cells previously studied in this laboratory (designated SRD-1 and SRD-2 cells) showed a marked overexpression of HMGCoA synthase mRNA (>30-fold) underall conditions of growth, suggesting that this mRNA is normally kept under tonic feedback suppression even in the absence of exogenous sterols and that this suppression is relieved in the mutant cells (13). When the SRD-1 and SRD-2 sterol-resistant cells were transfected with chimeric plasmids containing the LDL receptor promoter or the HMG-CoA synthase promoter fused to a reporter gene, they produced large amounts of reporter gene mRNA (13). In contrastto theresults with transfectedparental cells, the mutant cells failed to suppress transcription of the chimeric genes when sterols were added. Nevertheless, when the sterol regulatory elements in the chimeric plasmids were mutated, transcriptionwas markedly reduced (13). These results suggest that the sterol-resistant SRD-1 and SRD-2 cells retain a protein that binds to the SRE-1 and activates transcription but they have lost the ability to inactivate that protein in the presence of sterols. In the current studies we have used the sterol-resistant mutant cells to determine whether the posttranscriptional control mechanism for HMG-CoA reductase operates through the same gene that controls transcription. In particular, we have asked whether 25-hydroxycholesterol will accelerate degradation of HMG-CoA reductase in cells that areresistant to its effects on gene transcription.

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reaction was initiated by the additionof [l-14C]acetyl-CoA.Following a 10-min incubation at 37 “C, a 45-pl aliquot was removed from the mixture and added to a scintillation vial containing 0.25 ml of 6 N HC1. Vials were heated to 95 “Cfor 2h, and the amount of nonvolatile [“CIHMG-CoA formed was quantitated by scintillation spectrometry. Reaction rateswere determined tobe linear for time of incubation and amount of protein added. HMG-CoA synthase activity is expressed as nanomoles of [‘4C]acetyl-CoA converted to [I4C]HMGCoA/min/mg of cytosolic protein. The mRNAs for hamster HMG-CoA reductase and LDL receptor were measured by quantitative SI nuclease analysis of total cellular RNA as described previously (13).The mRNAs for hamster HMGCoA synthase, ribosomal protein S17, and thymidine kinase were quantified by primer extension analysis of total cellular RNA as described previously (7,13). S1 nuclease-resistant or primer-extended products were subjected to denaturingpolyacrylamide gel electrophoresis, dried, and exposed to x-ray film at -70 “C with intensifying screens. Quantification was performed by densitometry using a Hoefer scanning densitometer(model GS 300) or by radiometric analysis of dried gels using the Ambis Radioanalytic Imaging system (Ambis Systems, San Diego, CA). For immunoprecipitationof 35S-labeled cellextracts, thecells were washed twice and harvested in ice-cold phosphate-buffered saline containing 20 mM unlabeled methionine and1 mM unlabeled cysteine. Cell pellets were lysed in RIPA buffer (10 mM sodium phosphate at p H 7.5, 5 mM EDTA, 5 mM EGTA, 100 mM NaC1, 1% (v/v) Triton X-100,0.1%(w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1 mM phenylmethanesulfonyl fluoride, 20 p~ leupeptin, 1 mM dithiothreitol, 20 mM methionine, and 1 mM cysteine) by repeated aspiration through a 25-gauge needle. The extractwas centrifuged a t 10,000 X g for 5 min, and aliquots of the supernatant were removed for determination of protein content and trichloroacetic acid-precipitable radioactivity. For HMG-CoA reductase immunoprecipitation, aliquots of solubilized cell extracts (200-500 pg) were diluted 5-fold with fresh RIPA buffer and incubated with 10pg of polyclonal anti-HMG-CoA reductase IgG orpreimmune IgG for 1 h a t 4 “C. Theimmune complexes were incubated for 1 h a t 4 “C with 10 p1 of protein-A Sepharose (Pharmacia LKBBiotechnology), collected by centrifugation, washed five times with fresh RIPA buffer, and dissolved in 100 pl of SDS samplebuffer (21). For immunoblot studies of HMG-CoA reductase, cells were harvested in phosphate-buffered salineand lysed in buffer A (15% SDS, 8 M urea, 10% (w/v) sucrose, 62.5 mM Tris-HC1, and 5 mM dithiothreitol at pH6.8) by repeated aspiration through a 25-gauge needle. For immunoblot analysis of HMG-CoA synthase, the same dialyzed cytosolic fraction prepared for enzymatic assay (see above) was used. Immunoblotting was performed asdescribed (22) except that 5% (w/ v) nonfat dry milk was used in place of bovine serum albumin and 0.05% (w/v) antifoam A (Sigma) was included in the blottingbuffer. Quantification was performed by densitometryor by radiometric analysis of the nitrocellulose filter using the Ambis Radioanalytic Imaging System. RESULTS

Fig. 1shows the lack of suppression of HMG-CoA reductase activity by 25-hydroxycholesterol in a sterol-resistant mutant cell line (SRD-3) ascompared with the effect in the parental cells (Chinese hamster V-79 lung fibroblasts). The mutant cells showedno significant suppression at 4 lg/ml of 25hydroxycholesterol, which was 10-fold higher than the level that suppressed by 85% in the parentalcells. Fig. 2 explores the multivalent regulation of HMG-CoA reductase in the parental V-79 and mutant SRD-3 cells. In the V-79 cells mevalonate and 25-hydroxycholesterol each suppressed HMG-CoA reductase activity by more than 80%, and the combination was more effective than either alone (Fig. 2 A ) . When the V-79 cells were treated with compactin, the multivalent effects of sterols and mevalonate was amplified (Fig. 2B). HMG-CoA reductase activity rosebymore than y-fold, and there was only partial suppression by 25hydroxycholesterol or mevalonate. The two agents together reduced enzyme activity by a further5-fold. In theabsence of compactin, the SRD-3cells showed a blunted suppression by 25-hydroxycholesterol and by mevalonate and a more signif-

i n Sterol-resistant Cells

25-Hydroxycholesterol (pghnl)

FIG. 1. Suppression of HMG-CoA reductase activity in V79 and SRD-3 cells by 25-hydroxycholestero1. On day 0, cells were plated a t 3 X lo4cells/60-mm dish in 3 ml of medium A in the absence (V-79cells) or presence of 1 pg/ml25-hydroxycholesterol (SRD-3 cells). On day 2, all cells received 3 ml of medium A with no added 25-hydroxycholesterol. On day3, cells received 2 ml of medium A and the indicated concentration of 25-hydroxycholesterol. After incubation for18 h a t 37 “C, thecells were harvested for measurement of HMG-CoA reductase activity. The “100% of control” values for the V-79 andSRD-3 cellswere 229 and 447 pmol.min”.mg of protein”. Each value is the average of duplicate or triplicate incubations.

IA.

NO

V-79Cells I + Compactin IC.

Cornpactin B.

SRD-3 Cells ID.+ Compactin

NO Cornpactin

Additions to Medium

FIG. 2. Regulation of HMG-CoA reductase activity in V-79 ( A and B ) and SRD-3 cells (C and D ) cultured in the absence and presence of compactin. Cells were set up on day 0 in 60-mm dishesas described inthe legend to Fig. 1. Onday 3, each cell monolayer was refed with 2 ml of medium A in the absence ( A and C) orpresence ( B and D ) of 10 p~ compactin.Onday 4, after incubation for 18 h, each dishreceived fresh medium with or without compactin as indicated and one of the following additions: none, 2 pg/ml25-hydroxycholesterol (25-HC), 15 mM sodium mevalonate (Meu.), or a combination of both 25-hydroxycholesterol and sodium mevalonate (25-HC + Meu.). After incubation for 6 h at 37 “C, the cells were harvested for measurement of HMG-CoA reductase activity. Each value is the mean of triplicate incubations. The number above each bar denotes the percentof the “no addition”controls.

icant 44% suppression by the two together (Fig. 2C). When the SRD-3 cells were treated with compactin, HMG-CoA reductase activity rose as much as itdid in the V-79 cells (Fig. 2 0 ) . This increase was largely reversed by 25-hydroxycholesterol (78% suppression), only slightly by mevalonate (36% suppression) and more profoundly (90% suppression) by the two together. We next conducted a series of experiments to explore the mechanism for the regulation of HMG-CoA reductase activity in SRD-3 cells in the presence of compactin and to determine whether similar regulation occurred for other sterol-regulated genes. The results arepresented qualitatively in Figs. 3-5 and



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summarizedquantitatively in Table I. Fig. 3 demonstrates that the changes in activity of HMG-CoA reductase in the compactin-treated SRD-3 cells were associated with changes in the amount of HMG-CoA reductase protein without corresponding changes in the amount of mRNA. Protein levels were estimated by immunoblotting (row A ) , and mRNA was measured by quantitative S1 nuclease protection (row B ) . Whenthe V-79 cells were treatedwithsterols,reductase mRNA and protein both declined. In the presence of compactin the mRNA and protein were both induced, and this was reversed by sterols. A striking difference was observed in the SRD-3 cells. These cells had high levels of HMG-CoA reductase mRNA, and this level was not altered by treatment with compactin or sterols (row B ) . Despite the lack of change in mRNA,theamount of HMG-CoAreductaseprotein rose markedly in the presenceof compactin, and thiswas reversed by sterols (row A ) . Fig. 3 also shows that the amount of mRNA for HMG-CoA synthase was markedly elevated in the SRD-3 cells (row C). In contrast to its effect on synthase mRNA in the V-79 cells, compactindidnotincreasethis mRNA in the SRD-3 cells, and sterols did not suppress it. (Note that the untreated SRD-3 cells,like the previously reported SRD-1 and SRD-2cells (13),showed a greater than 30-fold increase in synthase mRNA as compared with the parental V-79 cells.) LDL receptor mRNA was also not regulated by compactin or sterols in the mutant SRD-3 cells

synthase protein in theV-79 cells as well as the SRD-3cells. When shorter exposures were made, we still did not observe any compactin-mediated change in the amount of HMG CoA synthaseproteinintheSRD-3 cells, andthisresult was confirmed by a failure to find a significant change in HMGCoA synthase activity (bottom row in Fig. 4). T o determine whether the compactin-mediated induction of HMG-CoA reductase protein in the SRD-3 cells was caused by an increased rate of synthesis, we pulse-labeled the cells for 50 min with "S-labeled amino acids and then isolated the radiolabeled HMG-CoAreductase by immunoprecipitation and quantified it by autoradiography (Fig. 5). In the V-79 cells compactin increased the rateof synthesis of HMG-CoA reductase, and thiswas reversed by sterols. In the SRD-3 cells the rate of synthesis of the enzyme was elevated, and there was no effect of sterols or compactin. Table I summarizes the quantitative data obtained from densitometric scanning of autoradiograms and from enzyme assays for the experiments shown in Figs. 3-5. In the SRD-3 cells the striking new finding is that compactincaused an 8fold increase in HMG-CoA reductase protein and a corresponding 6.7-fold increase in enzyme activity without any significant change in mRNA level nor in the rate of enzyme synthesis, and this effect was reversed by sterols. This posttranscriptional regulation also appears to take place in V-79 cells, but it is more difficult to appreciate because the superimposed transcriptional regulation that occurs in these cells. (row D). Thus, in the V-79 cells compactin increased the HMG-CoA The data of Fig. 3 suggested that compactin and sterols were affecting HMG-CoA reductase in the SRD-3 cells by a reductase mRNA and enzyme synthetic rate by 1.8- and 1.9of enzyme posttranscriptional mechanism. Inasmuch as HMG-CoA syn-fold, respectively, but it increased the amount thase does not apparently undergo posttranscriptional regu- protein and activity much more (5.8- and 4.3-fold, respectively). lation, we next determined whether compactin and sterols In the SRD-3cells the compactin-mediatedincrease in the had any effect on the amountof HMG-CoA synthase protein in the SRD-3 cells(Fig.4). In the V-79cells, HMG-CoA steady-state level of HMG-CoA reductase protein without any synthase mRNA, protein, and enzyme activity were all sup- significant increase inenzyme synthetic rate implies a reducof enzyme degradation. To test this possibility pressed by sterols andinduced by compactin. The SRD-3cells tion in the rate massively overproduced HMG-CoAsynthase mRNA and pro-directly, we performed a pulse-chase experiment in the SRDtein even in the absence of compactin. There was no change 3 cells (Fig. 6). After a 50-min incubation with "73-labeled in proteinor mRNA when compactinor sterols were present. amino acids, the cells were washed and switched to medium The immunoblot of Fig. 4 is overexposed in ordert o show the containing an excess of unlabeled methionine and cysteine. TABLEI Relative effect of compactin and sterols o n HMG-CoA reductase, HMG-CoA synthase, and LDL receptor i n V-79 and SRD-3 cells Cells were set up for HMG-CoA reductase activity measurements as described in the legend to Fig. 2. LDL receptor mRNA, HMG-CoA reductase protein and mRNA, and HMG-CoA synthase protein and mRNA were analyzed asdescribed in Figs. 3 and 4. For quantification, the relative amounts of mRNA productswere determined by densitometry or radiometric analysis and normalized in reference to the signal produced by ribosomal protein SI7 mRNA (or thymidine kinase mRNA insome experiments, Ref. 7). Each value represents the average of two to five experiments. The average HMG-CoA reductase activity for V79 cells grown in medium A alone was 309 pmol. min" .mg protein". The average HMG-CoA synthase activity for V79 cells grown in medium A alone was 540 pmol .min" .mg protein". Additions to medium: -, none; S, sterols containing 10 pg/ml cholesterol plus 1 pg/ml 25-hydroxycholestero1; C, 10 pM compactin; S + C, sterols plus compactin. All values for "relative activity" are related to values in V-79 cells that received no additions. -cell I;ne ~~

Gene product

Parameter

~~~~~

SRD-3

v-79 -

S

C

s+c

-

s+c

S

C

4.6 2.7 1.0 1.3 0.8

4.6 3.6 8.0 6.7

4.6 3.6

21.9 97.8 10.3

relative activity

HMG-CoAmRNA reductase 0.1 mRNA HMG-CoA synthase

LDL mRNA receptor

level synthesis Enzyme Protein level 1.0 activity Enzyme level Protein level activity Enzyme level

0.4

1.0 1.0 1.0

0.3 0.5 0.1

1.0 1.0 2.2 1.0 2.3

0.3 0.7 0.4

16.72.815.6 75.4

1.0

0.4

1.5

1.8 1.9 5.8 4.3 0.9

0.7 1.1 0.9

3.7 3.1 1.1

0.5 0.8 0.8

11.0

81.2 11.5

20.8 77.0 10.8

0.4

1.7

1.6 2.0

2.3

1.2

Posttranscriptional Regulation in Sterol-resistant Cells

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v-79

Compactin Sterols

Cornpactin Sterols

rnRNA

Protein

Activity D

LDL Receptor mRNA

SRD-3

p pw u -

FIG.3. Sterol-mediated regulation of HMG-CoA reductase, HMG-CoA synthase, and LDL receptor in V-79 and SRD-3 of compactin. On day cells grown in the absence and presence

[ . 2.2 1. I 3.4 0.8 20

18 23 24

FIG. 4. Regulation of HMG-CoA synthase mRNA, protein, and activityin V 7 9 and SRD-3cells. On day 0, cells were plated at 2.5 X 10"/100-mm dish in 8 ml medium A. On day 1, the cells were refed with medium A. On day 3, the cells received 8 ml of medium A containing 100 p~ mevalonate in the absence (-) or presence (+) of 100 p~ compactin. After incubation for 16 h, the cells received the same medium as on day 3 supplemented with a mixture of 10 pg/ml cholesterol and 1 pg/ml 25-hydroxycholesterol as indicated. After 18 h, the cells were harvested for measurement of total cell protein and RNA. Aliquots of total RNA (20 pg) were analyzed by primer extension analysis for HMG-CoA synthasemRNAas described under "Experimental Procedures." The gel was exposed to x-ray film for 3 h a t -70 "C with an intensifying screen. For analysis of HMG-CoA synthase protein, 30 pg of cytosolic protein was subjected to SDSpolyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with 7.2 pg/ml of rabbit anti-HMG-CoA synthase antibody. The antibody was detected using "."I-labeled goat anti-rabbit IgG (2 X lofi cpm/ml, 4500 cpm/ng). The nitrocellulose was exposed to xray film for 1 h at -70°C with an intensifying screen. Cytosolic HMG-CoA synthaseactivity was determinedas described under "Experimental Procedures" and is expressed as nanomoles of [''C] HMG-CoA formed/min/mg of cytosolic protein.

0, cells were plated a t 1 X lo5cells/lOO-mm dish in 10 ml of medium A. On day 2, the cells were refed with the same medium. On day 4, the cells received medium A in the absence(-) or presence (+) of 10 p M compactin. After incubation for 16 h, the cells received the same medium asonday 4 supplementedwith a mixture of 10 pg/ml cholesterol plus 1 pg/ml 25-hydroxycholestero1 as indicated. After 6 h, cells were harvested for measurement of total cell protein and RNA. Aliquots of the solubilized cell extract (40 pg of protein) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with 10 pg/ml of anti-reductase monoclonal antibody IgG-A9 (18).Bound IgG-A9 was detected using "'1labeled rabbit anti-mouse IgG (2 X 10" cpm/ml; 4500 cpm/ng). The nitrocellulosefilterwasexposed to KodakXAR film for16h a t -70 "C with an intensifyingscreen. Aliquots of total RNA (10-20 pg) were subjected to either quantitative S1 nuclease analysis for HMGCoA reductase and LDL receptor mRNAs or primer extension analysis for HMG-CoA synthase and ribosomal protein S17 mRNAs as described under "Experimental Procedures." The gels were exposed t o Kodak XAR film for 2-16 h a t -70 "C with an intensifying screen.

At various intervals the cells were harvested and the amount of labeled HMG-CoA reductaseprotein was estimated by immunoprecipitation, SDS-gel electrophoresis, and autoradiography. When the SRD-3cells were grown in the absenceof compactin, the labeled HMG-CoA reductase was rapidly degraded, less than 20% remained after 3 h (Fig. 6 A ) . There was no influence of sterols in the absenceof compactin (Fig. 6 A ) .When thecells were grown in thepresence of compactin, the rateof degradation was markedly slowed, more than 70% of the enzyme was intact at 3 h (Fig. 6B). When sterols were added, there was a 7-fold increase in the rate of HMG-CoA reductase degradation so that only about 10% of the enzyme remained a t 3 h (Fig. 6B). This change is almost exactly sufficient to explain 6-fold the decline in HMG-CoA reductase protein and enzyme activity when sterols were added to the compactin-treated SRD-3cells (see Table I). We described previously (13) two clones of sterol-resistant hamster cells obtained by mutagenesis of CHO-7 cells, a line of Chinese hamster ovary fibroblasts. Designated SRD-1 and SRD-2, these cells were obtained by treatment with nitrosoethylurea and y-irradiation, respectively. To determine the genetic relationship between these lines and the SRD-3cells that were used in the current studies, we performed a series of somatic cell hvhrirlixnt.inn mrperirnontc Tn allnw fnr colontion of hybrid cells from eachof the threelines, each linewas firsttransfectedwithplasmidscontaining genes encoding resistance to hygromycin B or a neomycin analogue (G418). Stable transformantswere isolated, and pairwise fusions were performedwith polyethylene glycol. Hybrid colonies were

205 c?

0 x

11697-

L

6645 -

FIG. 5. Synthesis of HMG-CoA reductase by V-79 and SRD3 cells. Cells were set up for experiments on day 0 a t 5 X lo5cells/ 100-mm dish in 10 ml medium A. Onday 2, the cells were refed medium A in the absence (-) or presence (+) of 10 p~ compactin supplemented with a mixture of sterols containing 10 pg/ml cholesterol plus 1 pg/ml25-hydroxycholestero1 as indicated. After 16 h, the cells were washed with phosphate-buffered saline and incubated with 10 ml of methionine and cysteine-free medium containing 10% newborn calf lipoprotein-deficient serum and the same additions as on day 2. After 45 min, the cells were refed with thesame medium containing 200 pCi/ml of Tran['"S] label and pulsed for 50 min without chase. Cell extracts containing 2 X 10' cpm of "'S-labeled protein were analyzed by immunoprecipitation and SDS, 7% polyacrylamide gel electrophoresis as described under "Experimental Procedures." The gel was exposed to Kodak XAR film for 24 h a t -70 "C. I , immune I&; P, preimmune IgG.

selected by growth inthe presence of a mixture of hygromycin B and (3418. Replicate plates were grown either in the presence or absence or25-hydroxycholesterol, and the numberof surviving colonies was determined. Fig. 7 shows the results of several fusion experiments in



9133

droxycholesterol. This was true whether the CHO cells contributed the hygromycin resistance gene or the G418 resistance gene. Thus, the mutations in all three cells are genetically recessive with respect to thewild-type. When each of the cell lines was fused with itself. the mutant phenotype was maintained, i.e. there were as many colonies in the presence of 25-hydroxycholesterol as in the absence. The SRD-1 and the SRD-3 cells did not complement each other, i.e. the majority of hybrids formed between these cells maintained the mutant 25-hydroxycholesterol-resistantphenotype. In contrast, SRD-2cells complemented the defect in both SRD-1 and SRD-3 cells. Fusion of the SRD-2 cells to either the SRD-1 or SRD-3 cells gave hybrid cells that had the wild-type phenotype (i.e. sensitivity to25-hydroxycholesI I” o i 2 3 0 1 ; ; terol). Time of Chase (hours) The data of Fig. 7 suggest that the SRD-2 cells have a FIG.6. T u r n o v e r of HMG-CoA reductase in SRD-3 cells. mutation in a different gene than the SRD-3 cells. AccordCells were set up for experiments on day 0 a t 5 X 10‘’ cells/lOO-mrn ingly, we set up an experiment to determine whether the dish in 10 ml of medium A. On day 2, the cells were refed medium A SRD-2 cells would differ from the SRD-3cells in their sensiin the absence orpresence of 10 p~ compactin as indicated. After 16 tivity to the posttranscriptionaleffects of 25-hydroxycholesh the cells were washed with phosphate-buffered saline and incubated with 10 ml of methionine and cysteine-free medium including the terol. A comparison of the data in Fig. 8 with that in Fig. 3 shows that the SRD-2 cells, like the SRD-3 cells, retained same additions as on day 2. After 45 min, the cells were refed with regulation. Thus, the addition of the samemedium containing 150 pCi/mlof Tran[:”S] label. Following theirsensitivitytosuch a 50-min pulse, the cells were washed once with phosphate-buffered compactin to the SRD-2 cells markedly increased the amount saline and incubated with medium A containing 300 p~ unlabeled of HMG-CoA reductase protein (row A ) in the absence of a (-) or presence (+) of 10 p~ methionine and cysteine in the absence change in mRNA levels (row B ) , and this was reversed by compactin plus a mixture of sterols containing 10 pg/ml cholesterol genes in the SRD-2 and 1 pg/ml25-hydroxycholestero1as indicated. After chasing for the sterols. Despite the fact that the mutant cells and SRD-3 cells differed, the SRD-2 cells also showed a indicated time, cell extracts (400 pg of protein) were analyzed by immunoprecipitation and SDS, 7% polyacrylamide gel electrophoresis failure of sterol-mediated regulation of the mRNAfor all as described under “Experimental Procedures.” The gel was exposed three sterol-regulated genes (row B, D,and E ) . This latter t o Kodak XAR film for 48 h a t -70 “C and quantifiedby radiometric

oo

No Cornpactin

Ambis analysis.

A

:E B

Protein

L

Is)

f

HMG CoA

3

SRD9

E

cL 4308 5951

4347

60

E

2479

319

B

r

294

FIG. 7. Genetic complementation analyses of SRD cell lines. Pairwise fusion of G418-resistant (G418’) and hygromycin-resistant (Hyg’) cell lines was performed as described under “Experimental Procedures.” The number at theupper right-hand corner of each box represents the number of experiments in which a fusion was performed. The denominatorrepresentsthetotalnumber of hybrid colonies observed (colonies observed in the presence of 700 pg/ml G418 plus 300 pg/ml hygromycin), and the numerator represents the number of 25-hydroxycholesterol-resistanthybrid colonies observed (colonies observed in the presence of 700 pg/ml G418, 300 pg/ml hygromycin, and 1 pg/ml 25-hydroxycholesterol).

F

I

517

I

mRNA

FIG. 8. Sterol-mediated regulation of HMG-CoA reductase, HMG-CoA synthase, andthe LDL receptor i n C H Oand SRD2 cells g r o w n i n t h e a b s e n c e a n d p r e s e n c eof compactin. On day 0, cells were plated a t 4 X lo5(CHO-7) or 6 X lo5(SRD-2)cells/ 100-mm dish in 10 ml of medium A. On day 2, the cells were refed withmedium A. Onday 3 the cells received 8 mlof medium A containing 100 p~ sodium mevalonateandone of the following additions as indicated none, a mixture of 10 pg/ml cholesterol and 1 pg/ml 25-hydroxycholestero1, 100 p~ compactin, or compactin and After incubation for 24 h, cells were harvested for measurewhich we used all three mutant cell lines plus the wild-type sterols. ment of RNA and protein. Aliquots of total RNA (10-40 pg) were CHO-7 cells. In order to eliminate possible artifacts resulting subjected to quantitative S1 nuclease analysis for HMG-CoA reducfrom drug treatment, each cell line was studied under two tase and LDL receptor mRNAs or to primer extension analysis for conditions: 1) when it carried the G418 resistance gene and HMG-CoA synthase and ribosomal protein S17 mRNAs asdescribed 9) whan it octrr;nrl thn hypomycin rooiotoncc gono. Each box Irnrlnr “Rvpnrimnntnl Prnonrlnrnc ” C n l c wnrn nvpnanrl tn KnrloL Y 4. R in Fig. 7 shows the totalcolonies surviving in thepresence of 5 film for 1-18 h a t -70 “C with an intensifying screen. Aliquots of proteinextract (90 pg) were analyzed for HMG-CoA 25-hydroxycholesterol (numerator) and the total colonies sur- solubilized reductase and HMG-CoA synthase protein by immunoblot analysis viving in the absence of 25-hydroxycholesterol (denominator). as described in thelegends to Figs. 3 and 4. Nitrocellulose filters were Fusion of CHO cells to any of the three mutant cells gave a exposed to Kodak XAR-5 film for 10-48 h a t -70 “C with an intenwild-type phenotype, i.e., the hybrids were killed by 25-hy- sifying screen.

9134


finding is in agreement with our previous results in the SRD2 cells (13). DISCUSSION

The current data reveal that sterols accelerate the degradation of HMG-CoA reductase in mutant Chinese hamster cells that fail to show sterol-dependent repression of gene transcription. Thus, the gene that is defective in the sterolresistant cells is not required for the regulation of protein degradation. The protein produced by this gene mayonly play a role in transcriptional control of promoters that contain sterol regulatory elements. The mutant SRD-3 cells provide a stable background in which to study posttranscriptional regulation since HMGCoA reductase mRNA levels do not change in response to sterol deprivation. The studies allowed a clear demonstration that HMG-CoA reductase degradation is slowed by about 7fold in the presence of compactin and that thiseffect can be reversed by 25-hydroxycholestero1plus cholesterol. The sterols accelerated degradation in this study without a requirement for exogenous mevalonate. However, the incubations were performed in the presence of only 10 p~ compactin, a concentration that is likely to permit the production of sufficient mevalonate to supply a nonsterol isoprenoid that acts together with sterols to accelerate degradation (1).In previous studies in which a much higher concentration of compactin was used(100 p ~ ) ,accelerated degradation did not occur unless the cells were supplied with exogenous mevalonate as well as sterols (3). We did not use the higher concentration of compactin in this study because it complicates the HMGCoA reductase assays, requiring a dialysis step before an accurate measurement can be made (3). The concentration of compactin that we used was sufficient to show a clear-cut effect of sterols on degradation, which was the purpose of this study. Because we did not use 100 p~ compactin, we also cannot draw conclusions about translational control in these mutant cells. When the SRD-3 cells were grown in the absence of compactin, the rate of degradation of HMG-CoA reductase was high and there was no change upon sterol addition (Fig. 6 A ) . Because of their resistance to feedback control, the SRD-3 cells chronically synthesize about 18-fold more cholesterol than theparental V-79 cells, as determined by incorporation of [14C]pyruvate(data not shown). Apparently, this endogenously synthesized cholesterol accelerates enzyme degradation, and this acceleration is relieved only when cholesterol synthesis is blocked by compactin. Accelerated degradation in the SRD-3 cells in the absence of compactin is further suggested by the observation that these cells have a low amount of HMG-CoA reductase protein relative to theirlevel of mRNA when compared with the V-79 cells (Fig. 3). Thus, in the control state the SRD-3 cells have a 3.1- to 3.7-fold increase in the amounts of reductase mRNA and enzyme synthetic rate when compared with the V-79 cells, yet the amounts of enzyme proteinand activity arenot elevated (Table I). Sterol-dependent acceleration of HMG-CoA reductase degradation is mediated by the complex membranous domain of the protein (23), which includes seven membrane-spanning regions (18)and which anchors the protein to themembranes of the nuclear envelope and endoplasmic reticulum (24). A soluble truncated version of HMG-CoA reductase that lacks the seven membrane-spanning regions is degraded slowly in

i n Sterol-resistant Cells cells, and the rate is not accelerated by sterols (23). Moreover, sterol-regulated degradation has been demonstrated for a fusion protein containing the HMG-CoA reductase membranous domain joined to Escherichia coli &galactosidase (25, 26). The seven membrane spanning regions of HMG-CoA reductase are similar in organization, although notin sequence, to rhodopsin and tocell surface receptors that interact with GTP binding proteins (1, 18). Whether HMG-CoA reductase interacts with such a GTP binding protein is unknown. Accelerated degradation of HMG-CoA reductase is also associated with an increase inthe sterol content of endoplasmic reticulum membranes, as indicated by increased labeling with the sterol-binding antimicrobial filipin (27). This increase may bethe trigger that accelerates degradation. The cellular site at which this degradation occurs is unknown. Another regulatory function of 25-hydroxycholesterol, the stimulation of acyl-CoA:cholesterol acyltransferase activity, is preserved in SRD-2 cells (13) and in SRD-3 cells (data not shown). This action, like the accelerated degradation of HMG-CoA reductase, is posttranscriptional and must be mediated by cytoplasmic factors that are different than the nuclear ones that mediate 25-hydroxycholesterol effects on transcription. With these two regulatory mechanisms now clearly separated, it should be possible to focus separately on the two classes of proteins that mediate the nuclear and cytoplasmic effects of sterols. Acknowledgments-We thank Edith Womack and Lisa Beatty for excellent help with the tissue culture work. Gloria Brunschede and Debra Noble provided excellent technical assistance. REFERENCES 1. Goldstein, J. L., and Brown, M. S. (1990) Nature 343,425-430 2. Brown, M. S., Faust, J. R., Goldstein, J. L., Kaneko, I., and Endo, A. (1978) J. Biol. Chern. 253,1121-1128 3. Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 263,8929-8937 A Brown, M. S., and Goldstein, J. L. (1980) J. Lipid Res. 2 1 , 505-517 J. L., Slaughter, C. A,, and Brown, M. S. (1986) J. Biol.

7. Siidhof, T. C., Russell; D. W., Brown, M. S.,.and Goldstein Cell 48,1061-1069 8. Smith, J. R., Oshorne, T. F., Goldstein, J. L., and Brown, M. S. (1990) J. Biol. Chem. 265,2306-2310 9. Osborne, T. F., Gil, G., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 263,3380-3387 10. Smith, J. R., Osborne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G. (1988) J. Biol. Chem. 263,18480-18487 11. Leonard, S., and Sinensky, M. (1988) Bmchim. Biophys. Acta 9 4 7 , 10111"

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12. Chang, T-Y., andLimanek, J. S. (1980) J. Biol. Chem. 2 5 5 , 7787-7795 13. Metherall, J. E., Goldstein, J. L., Luskey, K. L., and Brown, M. S. (1989) J . Bzol. Chem. 264,15634-15641 14. Chang, T-Y., and Chan C C Y (1982) Biochemistry 21,5316-5323 K. and 15. Goldstein, J. L., Basu, ' , ' ' Brown, M. S. (1983) Methods Enzyrnol. 98,241-260 16. Lachmann, P. J., Strangeways, L., Vyakarnam, A,, and Evan, G. (1986) Ciba Found. Symp. 119,25-57 17. Luskey, K. L., and Stevens,B. (1985) J. Biol. Chem. 260,10271-10277 18. Liscum, L., Luskey, K. L., Chin, D. J., Ho, Y. K., Goldstein, J. L., and Brown, M. S. (1983) J. Biol. Chem. 258,8450-8455 19. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1,327-341 20. Balasuhramaniam, S., Goldstein, J. L., and Brown, M. S. (1977) Proc. Natl. Acad. Sci. U. S. A . 74c, 1 A 9 1 - 1 A 9 5 21. Chin, D. J., Gil, G., Fau!st, J. R., Goldstein, J. L., Brown, M. S., and Luskey, K. L. (1985) Mol. Gel,1. Biol. 5 , 634-641 22. van Driel, I. R., Davis, C. G., Goldstein, J. L., and Brown, M. S. (1987) J. Biol. Chem. 262,16127-16134 23. Gil, G., Faust., J. R., Chin, D. J., Goldstein, J. L., and Brown, M. S. (1985) Cell 4 1, 24!3-258 24. Pathak, R. K. , Luskey, K. L., and Anderson, R. G. W. (1986) J . Cell Bid. 102,2158-2168 25. Skalnik D. G., Brown, D. A,, Brown, P. C., Friedman, R. L., Hardeman, E. C.: Schimke, R. T., and Simoni, R. D. (1985) J. Biol. Chem. 2 6 0 , 1 IyJqI - L lJ n n~ J'i 26. Chu,n. K. T.. Bar-Nun., S... and Simoni. R. D. (1990) J. Biol. Chem. 2 6 5 , 22d04-22010 27. Orci, L., Brown, M. S., Goldstein, J. L., Garcia-Segura,L. M., and Anderson, R. G . W. (1984) Cell 36,835-845

8.

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