Modulation Of 3-Hydroxy-3-Methylglutaryl-Coa Reductase By 15 Alpha-Fluorolanost-7-En-3 Beta-Ol. A Mechanism-Based Inhibitor Of Cholesterol Biosynthesis

The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15 alpha-fluorolanost-7-en-3 beta-ol, are described. The 15 alpha-fluorosterol is shown to be a competitive inhibitor of the lanosterol 14 alpha-methyl demethylase (Ki = 315 microM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15 alpha-fluorosterol is converted to the corresponding 15 alpha-fluoro-3 beta-hydroxylanost-7-en-32-aldehyde by hepatic microsomal lanosterol 14 alpha-methyl demethylase but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 15 alpha-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14 alpha-methyl demethylase-deficient mutant cell line, AR45, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14 alpha-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15 alpha-fluorosterol-treated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in HMG-CoA reductase protein is due to inhibition of enzyme synthesis, suggesting that the 15 alpha-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the 15 alpha-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms.

The chemical synthesis and metabolic characteristics of the lanosterol analogue, 15a-fluorolanost-7-en-3~-01, are described. The l5a-~uorosterol is shown to be a competitive inhibitor of the lanosterol l4a-methyl demethylase (Kt = 315 PM), as well as substrate for the demethylase enzyme. Metabolic studies show that the 15a-fluorosterol is converted to the corresponding l6a-fluoro-3~-hydroxylanost-7-en-3~-a~dehyde by hepatic microsomal lanosterol 14a-methyl demethylme but that further metabolic conversion to cholesterol biosynthetic intermediates is blocked by virtue of the 16a-fluoro substitution. When cultured cells are treated with the fluorinated lanosterol analogue, a decrease in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and immunoreactive protein was observed. However, when the lanosterol 14a-methyl de-methyla~-de~cient mutant cell line, ARM, is treated with the fluorosterol, no effect upon HMG-CoA reductase is observed. Thus, metabolic conversion of the sterol to its 32-carboxaldehyde analogue by the lanosterol 14a-methyl demethylase is required for HMG-CoA reductase suppressor activity. Measurement of HMG-CoA reductase mRNA levels in 15a-fluorosteroltreated Chinese hamster ovary (CHO) cells reveals that mRNA levels are not decreased by the sterol as would be expected for a sterol regulator of HMG-CoA reductase activity. The decrease in ~MG-GOA reductase protein is due to inhibition of enzyme synthesis, suggesting that the lba-fluorosterol reduces the translational efficiency of the reductase mRNA. Measurements of the half-life of HMG-CoA reductase show that, in contrast to other oxysterols, the ~~~-fluorolanostenol does not increase the rate of degradation of the enzyme. Collectively, these data support the premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms. The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; GC/MS, gas chromatography/mass spectroscopy; TMS, t r i~e t h y l s i l y~ CHO, Chinese hamster ovary; NOE, nuclear Overhauser effect; DLSP, delipidated fetal bovine serum. ductase (EC 1.1J.34) is the rate-limiting enzyme governing cholesterol biosynthesis and the synthesis of other isoprenoids in mammalian cells (I). It has been suggested that regulation of this important enzyme in cellular metabolism i s under the control of a multivalent feedback process which involves both end product sterol, as well as other non-steroidal mevalonatederived products ( 2 ) . Results to date demonstrate that steroldependent regulation of reductase activity i s due primarily to a transcriptional control process which affects the level of reductase mRNA (3,4), although less dramatic post-transcriptional control by sterols has been observed (5). Conversely, the non-steroidal regulation of reductase has been shown to involve post-transcriptional mechanisms which entail enhanced enzyme degradation and decreased translational effeiency of reductase message (6, 7). Thus, the differences between the two control processes are highlighted in terms of mechanism as well as affector molecule.
Recently there has been renewed interest as to whether cholesterol or some oxysterol precursor or product of cholesterol metabolism is responsible for the observed steroidal regulation of HMG-CoA reductase activity, Generation of regulatory oxysterols by means of a side pathway through epoxycholesterol has been proposed as an alternative to cholesterol as the natural regulator of reductase activity (8)(9)(10)(11). Similarly, oxysterol intermediates generated along the normal pathway of cholesterol biosynthesis have been shown to modulate the reductase in a manner consistent with their involvement in the regulatory process (12, 13). In the latter case, it has been demonstrated that 3~-hydroxylanost-S-en-32-a1 which arises during the course of lanosterol 14a-methyl demethylation can serve to regulate HMG-CoA reductase activity when carbon flux through the sterol pathway is abnormally high (13). Mechanistic studies have also demonstrated that accumulation of 3~-hydroxy~anost-S-en-32-a1 is favored over demethylated end product under conditions when lanosterol substrate concentrations are saturating (14-16), thus supporting the cellular obse~ations on metabolic formation with kinetic data. Similarly, it has been suggested that the level of this same or comparable intermediate may decrease under conditions when substrate pools favor carbon flow to cholesterol (17). Collective~y, these observations support the notion that accumulation o~3~-hydroxylanost-S-en-32-al in situ may be the result of a designed integrated regulatory process which serves to control endogenous cholesterol biosynthesis under normal physiological fluxes of carbon flow-through the sterol pathway.
The importance of the 3~-hydroxylanost-S-en-32-~ in the regulation of HMG-CoA reductase and a detailed understand-

22592
Modulation of HMG-Co ~e d u c t~e by F l u o~~~n o s t e n o l ing of its mechanism have been hampered, however, by inadequate experimental systems which allow one to monitor specific effects of the endogenously generated sterol metabolite. One approach which we have taken to solve this dilemma has been through the generation of cholesterol biosynthetic mutants which allow dissection of the process genetically (18). A second and equally important approach is to use synthetic substrates. The latter approach has suffered in the past, however, from the inherent problem that synthetic metabolites are ultimately converted to cholesterol as a consequence of normal metabolism. To circumvent this problem, we have designed and synthesized a lanosterol analogue which can only undergo limited metabolism by the lanosterol 14a-methyl demethylase of the cholesterol biosynthetic pathway, The compound described, l5a-fluorolanost-7-en-3~-ol, by virtue of its 15a-fluor0 substitution, is incapable of undergoing a complete demethylation cycle and subsequent conversion to cholesterol (19,201 (Scheme I). This inability to be converted to cholesterol makes the 15a-fluorolanosterol an ideal agent to study the regulatory properties and mechanism of oxylanosterol formation and control of HMG-CoA reductase activity in cultured cells.
In this report, we describe the metabolic characteristics of the 15a-fluorolanostenol and conditions where the fluorinated sterol is metabolized to its aldehyde analogue. Additionally, we demonstrate that the 15a-fluorosterol is an active suppressor of HMG-CoA reductase activity in wild-type, but not ~anosterol l~~-m e t h y l d e m e t h y l~e deficient, CHO cells. Most i~t e r e s t~~g l y , the regulat~o~ of HMG-CoA reductase by the fluorinated lanostenol in cell culture experiments is shown to be due to a post-transcriptional process analogous to nonsteroidal reductase regulators and not transcriptional control as would be expected for a sterol. These results demonstrate that cholesterol precursor sterols regulate HMG-CoA reductase and cholesterol biosynthesis by a mechanism distinct from that of cholesterol and oxycholesterol ~e~b o~i t e s . These findings bring into question the identity of the putative HMG-CoA reductase non-steroidal regulator which functions as a post-transcriptional modulator of reductase expression.
Metabolism of the 15n-fluorolanostero1 proceeds through aldehyde formation. Further metabolism of the lanostenol requires 15ol-proton loss which is prevented by the 15a-flUOrO substitution.
analyses required the following assay m~~c a t i o n .
Toluene stock solutions containing 1 pCi of ~24,25-'W]24,25-dihy~o~anost"8-en-3fl~ 01 substrate, 41.7-250 nmol, in addition to 0,50, or 100 nmol of 15afluorolanost-7-en-3fl-ol for each substrate concentration, were evaporated in the bottom of each assay tube under Nz to remove all toluene. To each tube 5.0 mg of Triton WR1339 was added in acetone to dissolve the sterols and then once again dried under Nz to form a detergent-sterol film in the bottom of the tube. Dimethyl sulfoxide, 50 pl, was added to each tube and vortex-mixed to suspend the detergent-sterol matrix followed by additions of 25 pl of 1.0 mM AY9944 (in a 10% propylene glycol) and 10 pl of 25 pM sodium cyanide. Two mg of rat liver microsomes prepared from male Spraque-Dawley rats that were fed a 3% cholestyramine diet (16) were added to each tube in 390 &I of a I00 mM KxP04, pH 7.4,O.l mM EDTA, 0.1 EIM dithiothreitol, 20% glycerol buffer. The microsomes and contents listed above were ~u i l i b r a~d a t 37 "C for 5 min and the assay initiated with 25 pl of a 20 mM NADPH/isoc~trate dehydrogenase generator mix (16). All other aspects of stopping the assay, saponification, extractions, and analysis by reverse-phase WPLC were as described (16).
Metabolism of 125 nmol of lanost-7-en-3P-01 or 15a-fluorolanost-7-en-30-01 were performed under the same conditions as described above with the exception that 24,25-dihydroIanosterol was omitted. The extracted nonsaponi~abie sterols were split in equal portions and derivatized by the two schemes listed below and analyzed by GC/ MS.
Deriuatization of Sterols for GC/MS Analysis-The C32-oxysterol products of lanost-7-ene-38-01 and Ea-fluorolanost-7-ene-30-01 are unstable during GC/MS analysis, since they decompose to their respective A8(24) unsaturated sterol under the high temperature cond~tions employed during analysis (21). Therefore, separate procedures are described for characterizing the 3&32-dioi sterols versus the 3~-hydroxy-32-aldehyde sterols by GC/MS. The first procedure requires that 3~-hydroxy-32-aldehyde sterols be reduced to a 32hydroxy group prior to derivation. The sterol sample was dissolved in 500 p1 of methanol at room temperature, and 5.0 mg of NaBH, was added and allowed to react for 3 h. Water, 2 ml, was then added to quench the reaction, and the sterols were extracted with 10 ml of petroleum ether. The petroleum ether extract was evaporated under Ns gas and the reduced sterols subsequently derivatized to their respective trimethylsilyl ethers. The reduced sterols were dissolved in 200 p1 of dry pyridine and 200 btl of "Tri-Si1 TBT" reagent (Pierce Chemical Co.), capped under N2 gas, and reacted overnight at 80 "C. The TMS reaction mix was then dissolved in 10 ml of petroleum ether and backwashed with 2 ml of 10% solution of sodium bicarhonate. The organic phase was removed and evaporated to dryness under Nz. The final sterol product was dissolved in 250 a1 of toluene, and 4.0-pl aliquots were analyzed by GC/MS as described beiow.
The alternate method for derivatizing 3@,32-&01 sterols as well as their respective substrates and demethylated products was accom-Irla-DNI, 0 2 NADPH 9 + plished by performing the "Tri-Si1 TBT" reaction as described here without NaBH. reduction. As above, the final derivative was dissolved in 250 pl of toluene and 4.0 p1 was analyzed by GC/MS. GC/MS Analysis of Sterols-Derivatized sterols were analyzed by GC/MS employing a Hewlett-Packard 5890 gas chromatograph equipped with 5970 mass selective detector (14). A capillary column of DB17-30W (J & W Scientific, Inc.) was used for sterol separations with helium carrier gas and the following temperature program: starting temperature, 235 "C; ramp rate, 5 'C/min; final temperature, 265 "C. Total ion chromatograms were run in the ion range of 50-800 atomic mass units. Cell Culture-CHO cells were cultured in McCoy's 5A medium (modified) supplemented with 1% Cab-0-Si1 (Kodak) delipidated fetal bovine serum (DLSP) as described previously (13). Cells were seeded at a density of 0.3 X lo6 in 60 X 15-mm tissue culture dishes (Costar) in 4 ml of the above medium 48 h prior to the start of each experiment (13).
AR45 cells, a CHO cell mutant deficient in lanosterol demethylase, were cultured in McCoy's 5A medium (modified) supplemented with 2.5% fetal bovine serum as described (18). Cells were washed twice with Hanks' balanced salt solution (GIBCO) before replacing the medium with 4 ml of McCoy's 5A medium (modified), 1% DLSP, 18 h prior to the start of experiment.
Determinatwn of HMG-CoA Reductase Activity, Immunoreactive Protein, and Quuntitation of mRNA Levels in Sterol-treated CHO Cells-HMG-CoA reductase activity and immunoreactive protein measurements were performed on cell homogenates exactly as described previously in detail (13) or on permeabilized cell cultures as reported by Leonard and Chen (22). Immunoblots were quantitated by scanning with an LKB UltroScan L densitometer.
HMG-CoA reductase mRNA levels were quantified by Northern blot analyses of poly(A+) RNA. For these determinations, cells were seeded in 150-mm tissue culture dishes (Costar) at 0.9 X 106/dish in 20 ml of McCoy's 5A (modified) medium containing 1% DLSP. Cells were allowed to grow for up to 5 days at 37 "C in 5% C02. On the day of treatment, the medium was replaced with fresh medium, and cells were treated with sterols suspended in 2.5% bovine serum albumin in 5% ethanol such that the final ethanol concentration did not exceed 0.5% (v/v). All treatments were done in triplicate. The treatment was continued for 16 h at which time cells were harvested by first removing culture medium and washing with 3 X 10 ml of cold (4 "C) 50 PM Tris-HC1, 155 pM NaCl, pH 7.4 (Tris/NaCl buffer). A small portion (14 cm') of each culture which was removed by scraping into 0.7 mf of Tris/NaCl buffer for activity measurements and immunoblots. Total RNA was isolated from the remainder of the cultures by guanidine isothiocyanate extraction and CsCl gradient centrifugation, with further fractionation by oligo(dT)-cellulose chromatography (23). Poly(A+) RNA (2.5 pg) quantitated spectrophotometrically was electrophoresed in 0.66 M formaldehyde, 1% agarose gels (23). Following transfer to nitrocellulose filters, hybridizations were performed with the following nick-translated [ C X -~~P J~A T Plabeled cDNA probes (23): the BamHI insert of the HMG-CoA reductase plasmid pRed227 (obtained from ATCC); the PstI insert of the @-actin cDNA (24); and the PstI insert of the gluteraldehyde phosphate dehydrogenase plasmid. pGAPDH plasmid (a kind gift from K. Hastings and C. P. Emerson, Jr. to P. Benfield, Du Pont) (25). The latter two probes were used as internal standards to quantitate mRNA recovery. Autoradiography at -70" for 3-24 h was used to detect the location of radioactivity on filters, and mRNA quantitation was done by cutting individual bands from the filter and counting in a liquid scintillation counter. Similar results were obtained by densitometric scanning of autoradiograms.
HMG-CoA Reductase Synthesis and Degradation-Exponentially growing cells were labeled in medium containing [35S]methionine (40 pCi/ml, 40 Ci/mmol). For synthesis measurements, cells were pulsedlabeled with [36S]methionine for 30 min at various times after treatment with sterols. For degradation measurements, untreated cells were labeled for 2 h with [35S]methionine and then cell monolayers were rinsed twice and incubated in medium containing 10 mM unlabeled L-methionine with or without oxylanosterol. Cells were harvested in Tris/NaCl and frozen at -80 "C. HMG-CoA reductase was immunoprecipitated essentially as described by Tanaka et al. (5). HMG-CoA reductase was immunoprecipitated with polyclonal antireductase igG. immunoprecipitates were solubilized at 37 "C in SDSurea sample buffer and analyzed by electrophoresis on SDS, 10% polyacrylamide slab gels [35S]methionine-labeled proteins were visualized by fluorography using EN3HANCE (Du Pont-New England Nuclear). Radiolabeled HMG-CoA reductase was quantitated by cut-ting the appropriate band from the gel, solubilizing the slice with Protosol (Du Pont-New England Nuclear), and counting in a liquid scintillation spectrophotometer. Background radioactivity was determined with preimmune rabbit serum. The half-life of HMG-CoA reductase was calculated from the slope of a semi-log plot of disintegrations/min in reductase versus time of chase.
Protein Determinations-Protein was determined by the Bio-Rad dye binding assay according to the manufacturer's directions employing bovine serum albumin as a standard.
Physical data (2) The reaction was stirred for 10 min, then quenched with the addition of sodium sulfate decahydrate (1 9). The mixture was diluted with diethyl ether (50 ml) followed by ethyl acetate (20 ml). The resultant solution was filtered though a sintered glass funnel and the solvents removed in vacuo. The resulting solid was crystallized from isopropanol to give 230 mg (96% yield) of 15a-fluorolanost-7-en-3P-ol (3). The crystals had a m.p. = 159-160 "C, and the NMR showed cocrystallization with isopropanol (2:1, 3:isopropanol). The crystals were then dissolved in benzene and the solvent removed to give an amorphous solid. Physical Data ( Synthesis of lanost-7-en-3P-01 used in these studies was performed as described by Woodward et al. (26).
Other Materials-A11 reagents used for lanosterol 14a-methyl demethylase and HMG-CoA reductase activity determinations were as described previously (13, 16 Schie~cher and Schuell, and x-ray film was from Kodak. Anti-HMG-CoA reductase IgG was a generous gift of Dr. Gene Ness, University of Southern Florida. All other reagents were the best grade commercially available.

C~r~t e r~z u t~n
of J5a-Fluorolanost-7-en-3~-ol-The 15a-fluor0 stereochemica~ assignment was critical for further studies with the l5a-ffuorolanosteno~ in our enzymic and cell biolog~ca~ studies. This assignment was based upon NMR studies. The ffuorine-pro~n coupling constant of 3.6 Hz which we observed between the C32-methyl and the ffuorine groups is consistent with the cis orientation as shown in Scheme I (31). However, these data alone were insuffic~ent for proper stereochemica~ assignment, A d~t i o n a~ evidence was also provided by a series of nuclear Overhauser effect (NOE) studies. Previous NOE experiments conducted on 3p-benzoyIoxytanost-7-en-l5icr-oi (27) gave an NOE between the 15s-hydrogen and the C18-methyl group. Conversely, the 38benzoyloxylanost-7-en-15fi-ol provided an observed NOE between the 15a-hydrogen and the 32 methyl. in the case of the only observed ff uorinated steroid from the ~e t h y~a m i n o sulfur triffuoride reaction, we observe an NOE between the 15-~y~o g e n and the 18-methy~ group, By analogy to the above cited experimen~, these NOE results, along with the ffuorineproton coupling cons tan^, allowed us to a s s i~e d the fluorinated steroid s t~c t u r e as the desired 3~-benzoyIoxy-l5a-ffuorolanost~7-ene (2).

Metabolic C~r~c t e r i s t~~ of I~a~F~~rolanost-7-en-38-nE with Hepntic
Microsomes-Once proper stereochemical assignments had been made, our initial studies with the 15afluorosterol were designed to assess its metabolic behavior with hepatic microsomes. Previously, WE have shown that lanostenols with a A7 double bond are poorly metabolized by the lanosterol demethylase despite having reasonable affiiity for the enzyme (32). This poor metabolic ~h a r a c~r i s t i c coupled with the inherent ~fficulty in detect~ng oxylanosteno~s made formation and d e~c t i o n of the 15a-ffuoro-3P-hydroxy-lanost~7-en-32-aldehyde a difficult task. Our efforts, therefore, did not focus upon detailed kinetics of formation, but rather upon simple demonstration of formation with more extensive characterization of the 15a-~uorolanos~roI by inhibition kinetics toward the lanosterol 14a-methyl demethylase.
Shown in Fig. 1 is the GC/MS chromatogram of reaction products generated during incubation of 15a-ffuoroianost-7en-3fi-01 with hepatic microsomes. The peak corresponding to  (Table I). This finding indicates that the Ilia-alcohol is present in the reaction mixture in much smaller amounts compared with the l~~-a l d e h y d e , again consistent with previous metabolic studies employing ~h y~o l a n o s t e r o l (21).
Evidence for me~bolism of the 15a-fluoroIanosterol to the 32-formyloxy interme~ate was not obtained in these studies. We a t t r i b u~ this lack of a~~~t y to generate the formylox~ intermediate to the inherent low rate of metabolism of the A7 lanosterol series (32) and the inability to generate the formyloxy intermediate when using lanosterol as substrate due to the kinetic properties of the 14a-demethylase enzyme (33).
Further eva~uation of the l5a-~uorolanostenol as a substrate fox the lanosterol 14a-methy~ demethy~ase was based upon inh~b~tion kinetics employing 24,25-~hy~olanosterol as substrate with a cell-free preparation of the demethylase enzyme. The double-reciprocal plot of demethylase activity in the presence of several conce~trations of the 15a-~uorolanost-7-en-3/3-01 is shown in Fig. 2. The data clearly demonstrate that the sterol is a competitive inhibitor of the demethylase with respect to dihydrolanosterol showing a IC, = 315 pM. These data are consistent with the observation that the 15afluorosterol inhibits the conversion of C30 sterols to C,, sterols in cultured CHO cells (50% inhibition at a concentration of 5 PM in the culture medium, data not shown). Thus, the notion that the 1 5~-~u o r o~a n o s t e n o~ is a substrate for the lanosterol Ilia-methyl d~methylase is confirmed by kinetic studies as well as by metabolic convers~on.

The Effect of ~~a -F~~~~~~s t -7 -e n -3 / 3 -0 1 upon ~~G~C o A ~e~~t~e Actiuity in Chinese ~u~t e r
Ovary Cetls-The next series of experiments with the fluorinated sterol were conducted in CHO cells. Our interest was in the ability of the compotrnd to modulate ~~G -C o A reductase activity through an oxys~rol-dependent mechanism. As shown in Fig. 3, the l5a-fluorol~nosterol shows a concentration-dependent suppression of ~M G -C o A reductase activity. This is a unique and specific property for the 15a-fluoro-substituted fanos-

TABLE I GC/MS c~~~~e r i~u t~o n of ~~~~l i t~s deriued from ~~s t -~-e n~3 B~ 01 and lfin-fluorokmost-7-en-3~-ol ~~~~~d with ~p u~i c
~~r o s o~~s I n c u~t i o n of sterols with hepatic microsomes was performed under standard lanosterol demethy~ase conditions for 1 h with the following exceptions: 250 phrf sterol, 5.0 mg of Triton WR-1339, and 10% dimethyl suIfox~de. Nonsaponi~able sterols were extracted as described previousiy (16) and were split into two equal portions for derivatization and analysis by the schemes outiined under "Methods." This ~i f f e~n t i a 1 derivatization procedure allows for quantitation of both Ianosteroi 14~-aicohol and 14n-aldehyde me~bolities, as well as demethylated diene sterol.  tenol, since lanost-7*en~~~-ol, the n o n -f l u o r~n~~d analogue, is without effect upon HMG-CoA reductase under identical conditions (Fig. 3). The nature of the suppression is consistent with an oxysterof-type ~e c h a n~s m since the decrease in ~M G -C o A reductase activity is accompanied by a decrease in the amount of immunoreactive reductase protein as shown in Fig. 4. Con~rmation that 15~"fluorinated lanosteno~ required metabolism by the lanosterol 14a-methyl demethylase to suppress HMG-CoA reductase activity in these cell experiments was obtained through investigations employing the ~anostero~ 14~-methy~ demethylase-de~cien~ m u t~t AR45 (18). Treatment of AR45 cells with the ~uorinated sterol does not produce a decrease in immunoreactive HMG-CoA reductase protein (Fig. 4B) nor does it inhibit reductase activity (control = 471 f 30 pmol/min/mg; sterol = 539 & 25 pmol/min/mg; n = 3). The lack of effect is not due to a nonresponsiveness of the AR45 cells to oxysterols, since suppression of HMG-CoA reductase is still observed when AR45 cells are treated with 25-hydroxycholesterol. Attempts to isolate the metabolic product of the 150-fluorolanosterol from wild-type CHO cul-tures have thus far been unsuccessful.

CoA Reductase Regulation by l5a-Fl~rolan~st-7-en-3~-ol-
To further characterize the suppression of HMG-CoA reductase caused by the 15a-fluorinated sterol, we measured the amount of reductase mRNA in treated and nontreated CHO cells. It was anticipated that the primary effect of the 15afluorosterol would be to change the level of reductase mRNA in a manner consistent with a transcriptional control process analogously to other sterols (3, 4). The effect with the 15afluorolanostenol upon reductase mRNA, however, was opposite to this exRectation. The results of a representative experiment are shown in Fig. 5 and Table 11. Chinese hamster ovary cells treated with the compound actually showed an increase in the amount of reductase mRNA compared with controls. In four independent experiments, the increase amounted to a 2-fold induction in reductase mRNA over a 16-h period. At the same time that the mRNA increase was observed, the level of reductase protein and activity decreased to 16% of control levels (Table 111). This effect appears to be unique for the fluorinated sterol since 25-hydroxycholesterol decreased the amount of reductase mRNA, protein, and activity in agreement with published results (3,4).
To further characterize the effects of the l5a-fluor0 analogue on HMG-CoA reductase gene expression, the rates of reductase synthesis and degradation were measured. Synthesis of HMG-CoA reductase in CHO cells was measured by monitoring the incorporation of [35S]methionine into immunoprecipitable protein in the presence or absence of sterol. The results of a representative experiment are shown in Fig.  6. Incubation of cells with the 15a-fluorolanostenol decreased the rate of synthesis of reductase (Fig. 6B) in the absence of any affect on total protein synthesis as assayed by the incorporation of [35S]methionine into acid-perceptible protein (Fig.  6A). Table 111,    CHO cells were treated with 25-hydroxycholestero1 (25-OH) or 15a-fluorolanosterol (15a-F). Enzyme activity, mRNA levels, and synthesis rates were determined as outlined under "Methods" and values are expressed as a percent of control. Enzyme half-life was determined as described in Fig. 6. All values are means f S.E., with the number of independent experiments given in parentheses.  (Table 111). It has been suggested that sterol regulation of reductase degradation is indirect and mediated by its effects on enzyme activity and mevalonate availability (7,34). Thus the lack of effect of l5a-fluorolanostenol on HMG-CoA reductase degradation might be related to its relative lack of potency as an inhibitor or reductase (Table 11). To test this possibility, 25-hydroxycholesterol, a potent transcriptional regulator of HMG-CoA reductase (Ref. 3 and Fig. 4), was added to cells at a concentration of 250 nM. This resulted in a 45% decrease in HMG-CoA reductase activity after 6 h, a value similar to that seen when cells are treated for 16 h with 10 p M 15a-fluorolanostenol. Under these conditions, 25-hydroxycholesterol increased the rate of degradation of HMG-CoA reductase 3.5-fold (data not shown). The inability of 15afluorolanostenol to accelerate reductase degradation is thus not a result of its more moderate inhibition of reductase activity. The data for oxysterol effects on HMG-CoA reductase activity, mRNA, synthesis, and degradation are summa-rized in Table 111. These results suggest that 15a-fluorolanostenol, in contrast to 25-hydroxycholesterol, is a post-transcriptional regulator of HMG-CoA reductase which acts solely at the level of translation.

DISCUSSION
The results presented in the current study characterize the metabolic behavior of 15a-fluorolanost-7-en-3~-01, a synthetic analogue of the natural substrate for the lanosterol 14amethyl demethylase enzyme. We have shown that the 15afluorolanostenol functions as a substrate for the lanosterol 14a-methyl demethylase, as well as serves as a good competitive inhibitor of the enzyme. Our results substantiate and extend previous findings that 15-fluoro-substituted lanosten-01s block cholesterol biosynthesis from lanosterol (30). By virtue of the 15a-fluor0 substitution, it has been possible to block complete metabolism of this lanosterol analogue to demethylated sterol which results in accumulation of oxygenated sterol under normal metabolic conditions. These findings indicate that 15a-fluor0 substitution in the lanostenol molecule is a modest modification which functions only to impede metabolic conversion to end product cholesterol, but not other properties of the lanostenol molecule. Thus, the ability to generate a stable oxylanosterol analogue through metabolism of the 15a-fluorosterol as described in this report provides us with another tool to study the mechanism of oxylanosterol regulation of cholesterol synthesis and HMG-CoA reductase activity.
Experiments in cultured cells with the 15a-fluorolanostenol have demonstrated that the 15a-fluorolanost-7-en-3P-01 is an active suppressor of HMG-CoA reductase activity under conditions when metabolism to an oxylanosterol analogue is permissive. We observe suppression of the HMG-CoA reductase activity only in wild-type CHO cells and not in the lanosterol 14a-methyl demethylase-deficient mutant, AR45. These results strongly suggest that metabolism of the 15afluorolanostenol by the lanosterol 14a-methyl demethylase enzyme is required for suppressor activity. Additionally, the decrease in enzyme activity is accompanied by a decrease immunoreactive protein which is consistent with previous reports on oxylanosterol regulation of HMG-CoA reductase activity (14).
Mechanistic studies with sterols, and in particular cholesterol and 25-hydroxycholesterol, have shown that sterol-de- 3sS]methionine for the indicated times in the presence of (0) or absence (0) of sterols. Incorporation of [%SI methionine into total acid-perceptible proteins and immunoprecipitable HMG-CoA reductase was determined as described under "Methods." A, acid-precipitable radioactivity. Data represent the average and standard error of duplicate determinations on replicate dishes. B , HMG-CoA reductase. Data represent disintegrations/min eluted from gel slices, corrected for nonspecific radioactivity. Curves were fitted by linear least squares analysis (Cricket Graph, Microsoft) (R2 > 0.9). C, CHO cells were pulse-labeled for 2 h with [35S]methionine and chased in the presence of unlabeled methionine for the indicated times. Cells were harvested and radiolabeled HMG-CoA reductase was quantitated as described under "Methods." Curves were fitted by exponential least squares analysis (R2 > 0.97). 0, ethanol control; 0, 15oc-fluorolanost-7-en-3~-01 was added at the beginning of the chase; W, cells were treated with 15a-fluorolanost-7-en-3~-ol for 16 h prior to the addition of label. pendent regulation of HMG-CoA reductase activity and expression is due to inhibition of transcription of the reductase gene (3). An octanucleotide cis-acting DNA sequence in ?ductme by Fluorolanostenol the 5"flanking region of the reductase gene appears to be responsible for sterol-dependent control of gene transcription and is thought to be the core binding site for a sterol-binding protein which represses transcription (35)(36)(37). It has also been demonstrated that regulation of reductase activity by sterols correlates with the ability of sterols to interact with a cytosolic oxysterol-binding protein (38). Viewed collectively, these data support the conclusion that sterols regulate HMG-CoA reductase through transcriptional control mediated at least in part through the action of a common oxysterol binding protein. The data we have presented, however, would indicate that this general conclusion regarding sterols may not be valid. Our results show that the 15a-fluorooxylanosterol analogue generated in situ regulates reductase expression through a post-transcriptional process. The decrease in reductase activity caused by the sterol is not accompanied by a decrease in reductase mRNA, but rather an actual increase. Similar post-transcriptional control of HMG-CoA reductase expression by 24,25-epoxylanosterol has been described (39). When metabolism of this compound to the epoxycholesterol was blocked, it no longer inhibited reductase activity in a rat intestinal epithelial cell line. However, in both primary rat hepatocytes and CHO cells, the oxidolanosterol post-transcriptionally inhibited reductase synthesis and enhanced reductase degradation even when its demethylation was inhibited with ketoconazole (39). In contrast to all other sterols examined to date, treatment of CHO cells with the 15afluorolanostenol has no effect on the degradation rate of HMG-CoA reductase. The 15a-fluorooxylanostenol metabolite thus appears to be unique in the manner of its regulation of HMG-CoA reductase, inhibiting translation without affecting either transcription or enzyme degradation.
The present findings dictate that previous assumptions and data used to support the concept of a non-steroidal regulator of HMG-CoA reductase activity be re-evaluated. In particular, the basic premise that all sterols regulate HMG-CoA reductase through a 25-hydroxycholestero1 mechanism is shown here to be false. Thus, experiments which employ 25-hydroxycholesterol to totally account for sterol regulation are subject to misinterpretation. For example, when 25-hydroxycholesterol is used in combination with mevalonic acid to regulate HMG-CoA reductase activity, the mevalonic acid effect cannot be assigned exclusively to a "non-steroidal" regulator. In fact, conditions which employ mevalonic acid to generate a non-steroidal regulator, either in mutant cells (6), or in combination experiments using 25-hydroxycholesterol plus compactin (7), favor synthesis of oxylanosterols (15). Of interest in this regard is the report that inhibition of HMG-CoA reductase by a 32-carboxylic acid derivative of lanosterol (SKF 104976) requires mevalonate (40), whereas inhibition by 24(S),25-oxidolanosterol does not (39). Additionally, Chin et al. (41) have shown that sterols can effect HMG-CoA reductase expression independently of transcriptional processes. Cohen and Griffioen (42) have also differentiated sterol metabolites of mevalonate whose effects upon reductase mRNA can or cannot be replaced by exogenous low density lipoprotein. Thus, the designation of the reductase translational regulator as non-steroidal should not be accepted without further proof of identity obtained through isolation and absolute characterization.
Our approach to understanding the mechanism of regulation of HMG-CoA reductase by endogenously generated oxysterols has focused on the lanosterol 14a-methyl demethylation cycle (13, 14, 16). Synthesis, chemical characterization, and metabolic studies with the novel substrate/inhibitor, 15afluorolanost"i-en-3~-ol, described in this report, extend our previous findings in this general area. The novel metabolic behavior of the 15a-fluorolanostenol demonstrates the possibility that manipulation of the lanosterol demethylation cycle through targeted synthesis can lead to a novel means to control HMG-CoA reductase activity and cholesterol biosynthesis. Our activities are continuing in this area.
A~~n o~~~g~e n t -W e thank Cathy Kieras for technical assistance, Dr. Gene Ness for supplying the anti-HMG-CoA reductase antibody, and Dr. Leonard G. Davis for assistance on molecular biological techniques.