THE JOURNAL. OF BiOLOGICAL ~ H E M I a T R ~ Q 1985 by The American Society of B~ologicai Chemists, Inc.

Yo\. 260. No. 27, Issue of November 25, pp. 14571-14579,1985 Printed in USA.

Identification of Regulatory Oxysterols, 2~(~,25=Epoxyc~o~estero1 and 25-Hydroxycho1estero1, in Cultured Fibroblasts"

(Received for publication, May 20,1985)

Sandra E. Saucier$, Andrew A. Kandutscht, Frederick R. Taylor$, Thomas A. Spencers, Seloka Phirwag, and Apurba K. Gayens From $The Jackson Laboratory, Bar Harbor, Maine 04609 and the $Department of Chemistry, Dartmouth College, Hwwuer, New Hampshire 03755

Bios~thetically tritiated sterols from Chinese ham- ster lung (Dede) cells were fractionated by high per- formance liquid chromatography, and fractions were assayed for their ability to repress 3-hydroxy-3-meth- ylglutaryl-CoA reductase in L cell cultures. Most o f the activity found was associated with two oxysterols, 24(~,25-epoxycholesterol and 25-hydroxycholes- terol. The identities of the two sterols were established by eo-chromatography with authentic samples and by isotopic dilution and recrystallization. Only low levels of repressor activity were found in other fractions of the sterol extract. The endogenous concentrations of 24(S),25-epoxycholesterol(7.2 fgfcell) and 25-hydrox- ycholesterol(l.5 fgfcell) appear to be within the ranges required for the regulation of HMG-CoA reductase.

Cultured cells respond to certain exogenous oxysterols, some of which are natural in~rmediates in steroid metabo- lism, by repressing the synthesis of HMGI-CoA reductase (1- 4). Derivatives of cholesterol hydroxylated in the 7a-, 26-, or 25-positions are produced in liver during bile acid production, (5, 6) , and side chain hydroxylation in the 20a- and 22R- positions is the first step in the conversion of cholesterol to steroid hormones in endocrine organs. In addition, all cells synthesizing cholesterol produce the obligate cholesterol pre- cursors, 32-hydroxylanos~rol and 32-oxo1~os~ro1, known repressors of the reductase (7,s). Recently, it has been shown that 24(S),25-epoxycholesterol is produced in liver, in vitro, by way of a branch in the sterol biosynthesis pathway begin- ning with the formation of squalene 2,3(S);ZZ(S),23-dioxide (9, 101, which is known to accumulate in cultured cells that have been treated with agents which block oxidosqualene cyclase (11, 12). The squalene dioxide is converted first to ~~(S),25-oxido~nosterol and thence to 24(S),25-epoxycholes- terol, which has been shown to be present in liver in vivo in a concentration about 10" relative to cholesterol? The mech- anism by which oxysterols repress HMG-CoA reductase ap- pears to involve a specific cytosolic binding protein, since the relative potencies of various oxysterols as repressors of the

* This work was supported by National Institutes of Health Grants CA 02758 and HL 23083. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: HMG, 3-hydrox~3-methylglutaric acid; DLSP, delipidated fetal bovine serum proteins; HPLC, high performance liquid chromatography.

Spencer, T. A,, Gayen, A. K., Phirwa, S., Nelson, J. A., Taylor, F. R., Kandutsch, A. A., and Erickson, S. K. (1985) J. Bid . Chem., in press.

reductase are closely correlated with their relative af~nities for the binding protein (1).

All of these findings lend support to a model for the regu- lation of the synthesis of HMG-CoA reductase by endogenous oxysterol metabolites complexed with the binding protein (1, 14, 15). However, further understanding of the regulation of HMG-CoA reductase requires more information regarding the number, identities, and concentrations of regulatory sterols in single cells. Therefore, in the present study we scanned the sterols o f Chinese hamster rung fibroblasts for their ability to repress HMG-CoA reductase activity. 32-Oxygenated sterols and 2~(S~,25-epoxysterols were considered to be kgical can- didates for a regulatory role in these cells because of their established natural occurrence (9-12): In fact, almost all of the repressor activity found was caused by just two oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol. The re- sults indicate that both of these sterols are produced in untreated, growing Chinese hamster lung (Dede) fibroblasts in concentrations sufficient to act as natural regulators of NMG-CoA reductase.

E X P ~ I ~ E ~ T A L ~ R O ~ E D ~ R E S Materials-~4(S),25-Epoxy[23-3H]cholesterol was prepared as de-

scribed previously? Unlabeled 24(S),25-epoxycholesterol was pre- pared as described pr~viously (9, LO), except that medium pressure liquid chromatography rather than preparative TLC was used to separate 24(S),25-epoxycholesterol benzoate from its 24(R) epimer. From 136 mg of a mixture of 24(S)- and 24(R),25-e~xycholesterol benzoate was obtained 49 mg of the S epimer and 55 mg of the R epimer by repeated application of 30-mg portions dissolved in 1 ml of benzene onto a column of 50 g of 230-400 mesh Silica Gel 60 and elution with 1.5:98.5 ethyl acetate:hexane, using a uv detector at 254 nm. Eluted Gactions were analyzed by TLC on 20 X 20-cm LK5D plates, using three develop men^ with 1.598.5 ethyl acetate:he~ne, which gave RF = 0.29 for 24(S),25-epoxycholesterol and RF = 0.26 for the R epimer. The separated 49 mg of 24(S),25-epoxycholesterol benzoate was saponified by treatment with 5 ml of 5% ethanolic potassium hydroxide solution at room temperature for 3 h. After solvent removal in vacuo, the residue was partitioned between ether and water. The organic layer was washed with water and brine, dried over magnesium sulfate, and evaporated to give a product which was purified by preparative TLC on a 20 cm X 20 cm X 1-mm plate of Silica Gel 60, PF,+,, eluting with 1:4 ethyl acetakhexane to afford 39 mg of solid which was recrystallized twice from acetone to give 24 mg of 24(S),25-epoxycholesterol, m.p. 158-160 "C (m.p. (literature)

24(S),25-Oxidolanosterol and 24(S),25-0xido~~H]lanosterol were prepared and fully characterized by procedures which will be de- scribed subsequently? 25-Hydroxy[26,27-3H]cholesterol (87 Ci/ mmol) from New England Nuclear was purified by TLC as described (1). The sources and methods of purification of other sterols were those described previously (1, 16, 17). Solvents for HPLC were from

' F. R. Taylor, A. A. Kandutsch, A. K. Gayen, J. A. Nelson, S. S.

160-162 "C (10)).

Nelson, S. Phirwa, and T. A. Spencer, manuscript in preparation,

14571

14572 Regulatory Oxysterols in Cultured Cells Burdick & Jackson Laboratories, Inc. Gentamycin sulfate, dl-a-to- copherol and linolenic acid were from Sigma. Tissue culture media were from K. C. Biological Inc. and fetal bovine serum was from Gibco Laboratories. (R,S)-[5-3H]Mevalonolactone and (R,S)-3-hy- dro~y-3-methy1[3-~~C]glutaryl-CoA were from New England Nuclear.

Cells-Chinese hamster lung (Dede) cells were grown at 37 "C in an atmosphere of 5% C02-95% air as monolayers in Corning Plastic flasks in McCoy's 5a medium supplemented with gentamycin sulfate (80 mg/liter), dl-a-tocopherol (0.5 pg/ml) and 3.6 mg/ml of DLSP. L cell cultures, used in assays for the repression of HMG-CoA reductase, were grown in modified serum-free Waymouth's 752/1 medium as described previously (1). Dede cell counts were made with a hemo- cytometer after release of the cells from the flask by trypsinization.

Delipidation of Fetnl Bovine Serum-The procedure used to remove all lipids capable of inhibiting HMG-CoA reduct? was a modifica- tion of the method of Rothblat et al. (18) for delipidating serum. Serum (100 ml) was slowly added to 10 volumes of 1:l acetone:ethanol with stirring at 0 "C, and the mixture was allowed to stand at 0 "C for 4 h. The serum proteins were sedimented by centrifugation at 500 X g for 10 min at -10 "C. The protein sediment was further extracted by stirring it first with 500 g of ether (anesthesia grade) previously chilled to -15 "C, then twice with 500-g volumes of anhydrous ether at -15 "C, centrifuging as before to sediment the protein, and finally with 600 ml of acetone at -15 "C. The acetone suspension was filtered on a Buchner funnel and the protein cake was dried in a vacuum dessicator which was repeatedIy evacuated over a period of 2 days until the cake was dry. The cake was then ground to a fine powder with a mortar and pestle.

To test whether oxygenated sterols were completely removed by this procedure, 1 pCi (2 pg) of 25-hydroxy[3HJcholesterol and 5 pg of butylated hydroxytoluene were added to 5 ml of fetal bovine serum, and the mixture was incubated for 3.5 h at 37 "C with gentle shaking, then overnight at mom temperature. The mixture was then delipi- dated as described above, and radioactivity in the various extracts and in the final DLSP preparation was determined. After the second ether wash 99.6% of the radioactive sterol had been extracted from the protein, and after the acetone wash only 0.1% of the initial radioactivity remained with the final DLSP preparation. As described under "Results," HPLC of a 2 1 CHC4:CHsOH extract of a DLSP preparation indicated the absence of cholesterol and of any activity in the HMG-CoA repression assay.

A stock solution of the delipidated serum protein was prepared by dissolving the powder in McCoy's medium at a concentration of 40 mg/ml. Linolenic acid and a-tocopherol in ethanol solution were added to a concentration of 0.85 &ml and 5.0 pg/ml, respectively (final ethanol concentration, 5 pl/ml), and the mixture was sterilized by filtration. The culture medium was prepared by diluting 1 ml of the stock DLSP solution with 10 ml of McCoy's medium.

Incubation of Cells with PH]Mevalonate and Extraction of the Sterol Fraction-Throughout the following procedures special care was taken to prevent auto-oxidation of sterols, acid-catalyzed hydrolysis of any epoxides, and trace contamination by radioactive or radioinert sterols present in the environment. Experiments were completed as quickly as possible, utilizing glassware that was either new or acid- washed. The glassware was rinsed thoroughly with distilled water, then once with dilute NH40H, and dried. Routinely, butylated hy- droxytoluene at a concentration of approximately 1 pg/ml was added to extracts and column fractions. A smali crystal of K&03 was added to extracts or fractions thereof before they were stored at -20 "C.

Dede cells were grown in 9-12 150-cm2 culture flasks until they approached confluency. They were then refed with 10 ml of fresh mediumlflask containing 100 pCi (10.5 pg) of [3H]mevalonolactone and incubation at 37 "C was continued for approximately 17 h. The cultures were then washed with 5 ml of cold (4 "C) McCoy's medium and scraped into the same medium. The pooled cells were sedimented in a 40-ml centrifuge tube at 200 X g for 5 min and suspended in 0.14 M NaCl, and aliquots were taken for protein determination and for the assay of HMG-CoA reductase activity. The remainder was cen- trifuged and the cell pellet was extracted with 20 volumes of 2:l CHC13:CH30H containing butylated hydroxytoluene and a crystal of KzC03. The addition of 0.03 M M&12 (0.2 volume) to the extract caused the separation of phases and the lower phase was evaporated under N2. The extract was taken up in 2 ml of 19:l hexane-CHCh and 1 ml was loaded onto each of two Sep-Pak siIica cartridges (Millipore, Waters Chromatography Division). Non-polar neutral lipids were eluted by passing 10 ml of 191 hexane:CHC13 through each cartridge and then the sterol fraction was eluted with 10 ml of

acetone. The acetone was evaporated under N2, and the residue was dissolved in 2 ml of methanol containing butylated hydroxytoluene. An aliquot was assayed for 3H and the remainder was filtered through a 0.45-p ACRO LC3S filter (Gelman Sciences) preparatory to HPLC.

The same procedures were also applied to cultures that had not been incubated with [3H]mevalonolactone. In one such experiment, 10 ng (9,250 dpm) of 24(S),25-epo~y[~H]cholesterol and 50 pg (20,425 dpm) of 25-hydro~y-[~H]cholesterol were added to the pelleted cells to allow estimations of recoveries.

HPLC and Gas Chromatography-HPLC was performed at mom temperature with a Waters HPLC instrument using: 1) a 10-p particle size, reverse phase, CIS semipreparative column 7.8 X 30 cm (Alltech Associates); 2) a 3.9 x 30 cm, IO-p particle size pPorasil column (Millipore, Waters Chromatography Division); and 3) a 5-r spherical, CIS Resolve column, 3.9 X 15 cm (Waters). Column eluates were monitored at 210 nm with an Isco Variable Wavelength spectropho- tometer; fractions were collected manually. Gas chromatography of sterols was at 285 "C on a 6-ft column of 4% OV 101 on Anakrom Q (Analabs, Inc.) with a Hewlett-Packard 5830A instrument.

Assays for the Repression of HMG-CoA Reductase in L Cell Cul- tures-Fractions obtained by HPLC were dried under N2, and the residue was dissolved in ethanol. Aliquots (usually 4) ranging from 2 to 20% of the total were added to 45 pl of 5% bovine serum albumin, and the concentration of ethanol was adjusted to 10%. The mixture was diluted with 0.45 ml of L cell medium and fed to L cell cultures prepared on the previous day by plating 3 X 1@ cells in 16-mm wells. Five hours later the cells were scraped into the medium, sedimented by centrifugation in conical cryotubes (Nunc) and the pellets were frozen in liquid Nz. To assay HMG-CoA reductase activity, the pellet was homogenized in 30 pl of 50 PM potassium phosphate buffer, pH 7.4, containing 20 pM dithiothreitol and 1 mM EDTA by pumping it up and down in a 30-pl Micro/pettor syringe (SMI). An aliquot (12 pi) was taken for protein determination, and another aliquot (12 pl) was incubated with the reaction mixture in a total volume of 50 pl. Incubation conditions and the method for determining the [3H] mevalonate product were as described previously (19). 24(S),25-Epox- ycholesterol and 25-hydroxycholesterol concentrations in HPLC frac- tions were determined from a standard plot of the log of the concen- tration of 25-hy~xycholesterol or 24(S),25-epoxycholesterol against the activity of HMG-CoA reductase (Fig. 1, panel A). Repressor activity not conclusively associated with a sterol of known identity is expressed as units of activity, where one unit equals the activity of 1 ng of 25-hydroxycholestero1. Under the conditions of the assay 3 ng of 25-hydroxycholestero1 was sufficient to repress HMG-CoA reduc- tase activty by 50%.

Uptake of Exogenous Sterols by Dede Cell Cultures-Conditions for incubation of 25 cm2 Dede cell cultures with exogenous [3H]sterols were similar to those described above for the incubation of sterol fractions with L cell cultures, except that the culture medium was McCoy's 5a containing DLSP and other additions as described above. After incubation at 37 "C the cultures were washed four times with 5-ml volumes of protein-free McCoy's medium. The cells were then scraped from the flasks, sedimented by centrifugation, washed once with 0.14 M saline, and dissolved in 0.2 ml of 1 N NaOH. The solution was then diluted to 1 ml with H20, an aliquot was assayed for protein,

3H. and another aliquot was neutralized with 2 N HCl and assayed for

Assay for Binding to the Oxysterol Biding Protein-Aliquots of ethanol solutions of HPLC fractions (usually 0.5 to 4% of the total) were assayed for their ability to displace 25-hydro~y[~H]cholesterol from the oxysterol binding protein. Preparation of the cytosolic oxy- sterol binding protein from L cells, conditions for binding of the ligand and assay hy sucrose density gradient centrifugation were as described previously (1). Concentrations of oxysterols were deter- mined from a standard plot of the displacement of 25-hydro~y[~HJ cholesterol as a function of the concentration of unlabeled 25-hy- droxycholesterol or 24(S),25-epoxycholeste~l (Fig. 1, panel B).

Other Assays-Total cellular protein was determined by the method of Lees and Paxman (20). Soluble proteins were assayed by a dye binding assay (21) using reagents from Bio-Rad. 7-Dehydrocho- lesterol was quantitated by measurement of Asla.

RESULTS

The results shown below in Figs. 2,3, and 4 represent data obtained with four individual sterol preparations from Dede cell cultures. The cultures and the procedures employed to

FIG. 1. Standard curves for repression of HMG-CoA redue- tase in L cell cultures (A) and for binding to the oxysterol ~ i n d ~ ~ p r o t e ~ (B) by 24(S),25-e~xycholesterol(O) and 25- hydroxycholesterol (x). Points for each curve represent results of two assays.

obtain and fractionate the four sterol preparations were sirn- ilar except that two of the preparations were from cultures that had been incubated with [3H]mevalonolactone and two were from unlabeled cultures. The unlabeled sterol prepara- tions were used to determine the distribution in chromato- graphic subfractions of activity measured by the HMG-CoA repressor assay. These assays required the util~ation of large portions of the &actions, since, in many cases, it was necessary to repeat assays over a wider or narrower range of aliquots in order to obtain reasonably accurate estimates of repressor activity. The radiolabeled sterol preparations were used to establish the metabolic origin of sterol bands and the identi- ties of the bands by co-chromatography and co-crystallization experiments. HPLC AzIo elution patterns obtained with the four ateroi preparations were essentially superimposable and each of the figures, 2 through 4, represents combined results obtained with two preparations.

~ r ~ t ~ ~ ~ o n of Cellular Sterols OR a ~ e ~ ~ r e ~ ~ ~ ~ e Cx8 Reuerse Phase Column-Fig. 2 shows that the greatest amount of activity in the HMG-CoA reductase repression assay was found in a broad fraction (number 61, with a retention time of 13-22 min, which included all of the oxysbrol standads. Much smaller amounts of repressor activity were found in fractions 9,10,12, and 14, intermediate between the retention times of the monooxygenated sterols and the &oxygenated sterol standards. The large A,,, band in fraction 10 had a uv spectrum characteristic of 7-dehydrocholestero1; its retention time coincided with that calculated for cholesta-5,7,24-trien- 38-01 (22) using cholesterol and cholesta"5,24-dien-3~-01 as reference ~ m ~ u n d s . The authentic triene was not available for compa~~sons, and the sterol in fraction 10 has not been further characterized. As shown in Fig. 2, no distinct Azlo band co-chromatographed with 7-dehydrocholesteml. Most of

the radioactivity in the chromatogram (-85%) was associated with bands containing cholesterol and lanosterol; a minor fraction of the total radioactivity, -l%, was associated with the active band in fraction 6.

To test the possibility that some of the repressor activity shown in Fig. 2 may have originated from the delipidated serum added to the culture medium or by auto-oxidation of cellular sterols during extraction and fractionation, the fol- lowing control experiments were conducted. DISP serum (320 mg), equivalent to the amount present in the culture media used in Fig. 2, was extracted, and the extract was fractionated following the procedure used for the cultured cells. Azzo mea- surement did not show any band corresponding to cholesterol (lower limit of detection, -2 pg) and no repressor activity was found in any of the eluted fractions, indicating that .the activity found in F1g. 2 was not derived from the culture medium. The amounts of the principal sterols in the Dede celi extracts chromatographed in Fig. 2 were determined by gas chromatography to be: cholesterol, 3600 Fg; desmosterol, 34 gg; lanosterol, 0.6 pg. The mount, of A5~7-sterol estimated from the Azal.s of fraction 10 was 4 sg. Corresponding amounts of these sterols, freshly purified by recrystallization and HPLC on a Cls reverse phase semipreparative column, were combined, dissolved in CHCl&H@H, and treated exactly as the cell extracts illustrated in Fig. 2. 7-Dehydrocholesterol was used as a substitute for the A5*7-sterol in fraction 10. No activity was found in the polar (oxysterol) fraction, number 6 (retention time, 13-22 min), indicating that the activity found in this fraction in Fig. 2 did not arise from a u t o - o x ~ ~ ~ o n of the principal sterols in the cell extract during processing. However, some repressor activity (320 units) was detected in fraction 10. This value may be compared with 750 units in fkaction 10 of the cell extract in Fig. 2.

~ ~ c ~ r o ~ t o g r ~ ~ y of the Cellular Polar (Oxysteroll Fruction on pPormiZ-Fraction 6 from the semipreparative CIS reverse phase column (Fig. 2) was dried under Nz, redissolved in 0.2 mi of 1.5% isopropanol in hexane, and injected onto a FPorasil column. As shown in Fig. 3, two major bands of activity in the HMG-CoA repression assay with the retention times of 2~(S~,25-epoxycholesterol (6.7 min, fraction 6) and 25-hy- ~oxycholesteroi (15.4 min, fraction 15) were eluted. These bands were correlated with peaks of radioactivity and with A Z ~ O peaks.

In addition, a very low level of repressor activity was detected in fractions 2-4 and a low level of repressor activity was eluted in fractions 20 and 21 after the proportion of isopropanol in the solvent mixture w8s increased from 1.5 to 3% in order to elute any 7-ketocholesterol or 7b-hydroxycho- lesterol. 7-KetocholesteroI and 7~-hy~oxycholeste~l are not thought to be normal metabolites, but they are major products of cholesterol auto-oxidation (23). To determine whether any of the labeled sterol in fractions 20 and 21 was 'I-ketocholes- terol, 31.5 mg of the authentic sterol was added to the pooled fractions (4482 dpm) to give a specific activity of 142 dpml mg. After three recrystallizations from acetonitrile the specific activity had declined to 31 dpm/mg, indicating that less than 22%, if any, of the radioactivity was associated with ?-keto- cholesterol. This result, along with the absence of appreciable radioactivity, Azla, or repressor activity in fraction 27, which would include any 7@-hydroxycholesterol, provided further evidence that cholesterol auto-oxidation was effectively inhib- ited under the conditions employed.

Lanosterol derivatives oxygenated at C-32 were not avail- able, so the corresponding 24,25-dihydro compo^^ had to be used as standards to approximate the expected retention times on pPorasil. An isolated double bond usually bas little

14574 Regulatory Oxysterols in Cultured Cells

L

lO t i31S3 lOH30M0AH30-L -

10ti31s0ws30 -

~lOM31SONVlOXO-M;.,- l (W31S310H3013M -L -

lOM31SONVl001XO-SZ '(S)+Zr

Regulutory Oxysterok in Cultured Cells 14575

l O Y 3 1 S 3 1 0 ~ 3 0 1 3 X - L-

l O Y 3 l S 3 l O W 3 W O ) - S l -

14576 Regulatory Oxysterols in Cultured Cells

effect in this chromatographic system, so we are confident that the dihydro derivatives provide a reasonably accurate guide to the actual retention times. No activity in the HMG- CoA reductase repression assay was detected in the region of the chromatogram containing lanost-8-ene-3/3,32-diol and only a trace of activity was located near the elution position of 3~-hydroxylanost-7-en-32-al.32-Oxygenated lanosterol de- rivatives are moderately effective repressors of HMG-CoA reductase (2-3 ~ L M for 50% repression) and exhibit corre- sponding affinities for the oxysterol binding protein (1, 7). The presence of about 5 pg (40 ng/mg protein) of either of these sterols in the cell extract would have given a readily detectable response in the assay for repression of the reduc- tase.

A trace of repressor activity and considerable radioactivity were also found near the elution position of 24(S),25-oxido- lanosterol, the putative biosynthetic precursor of 24(S),25- epoxycholesterol. Reverse phase HPLC (Resolve column) of pooled fractions 4 and 5 (Fig. 3) showed an Azlo peak and a corresponding radioactive peak with the retention time of 24(S),25-oxidolanosterol (data not shown). On the basis of the Azlo measurement in comparison with authentic 24(S),25- oxidolanosterol, the maximum possible amount of oxidolan- osterol was estimated to be 0.5 pg (4 ng/mg protein), and the associated radioactivity represented 7% of that applied to the column. The concentration of 24(S),25-oxidolanosterol re- quired for 50% repression of HMG-CoA reductase in L cell cultures was determined to be 0.68 PM: similar to the value of 0.89 p~ for 24(S),25-epoxycholesterol. It can be estimated from the value of 0.68 FM that there cannot have been more than about 1.5 pg (12 ng/mg protein) of the oxidolanosterol in the total cell extract or a clearly defined peak of repressor activity would have been observed. We have also found that 24(S),25-0xido[~H]lanosterol is rapidly metabolized to 24(S),25-epoxycholesterol upon incubation with Dede and L cell cultures, and that 24(S),25-oxidolanosterol does not bind detectably to the oxysterol binding protein: so that the con- tribution to regulation of cholesterol synthesis by this partic- ular oxysterol appears to be insignificant, at least in cultured fibroblasts.

Recrystallization of the Radwactive Bands in Fractions 6 and 15 (Fig, 3) with Authentic 24(S),25-Epoxycholesterol and 25-Hydroxycholesterol, Respectively-Fraction 6 (Fig. 3) was dried under Nz and dissolved in methanol. An aliquot was assayed for 3H, and 23.73 mg of unlabeled 24(S),25-epoxycho- lesterol was added to the remainder (85,200 dpm) to give a specific activity of 3,590 dpm/mg. Repeated recrystallization of this sterol established that the material from fraction 6 was indeed 24(S),25-epoxycholesterol, as the data in Table I in- dicate. Further confirmation was obtained by lithium alumi- num hydride reduction of a portion of the recrystallized epoxide having a specific activity of 2,970 dpm/mg. A solution of 6.8 mg (0.17 mmol) of this epoxide in dimethoxyethane was treated at reflux with excess LiALH4 according to our previous procedure,2 to afford, after purification by preparative TLC, 3.6 mg (53% yield) of solid which co-migrated with 25-hy- droxycholesterol. Recrystallization twice from acetone gave 25-hydroxycholesterol, specific activity 3,000 dpm/mg, m.p. 176-178 "C (m.p. (literature) 181.5-182.5 "C (24); 177-179 "c (25)). HPLC on a PPorasil column showed coincident absor- bance and radioactivity peaks with the retention time of 25- hydroxycholesterol (data not shown).

Fractions 15 and 16 containing 23,800 dpm were pooled, dried, and redissolved in methanol. Pure 25-hydroxycholes- terol (30.00 mg) was added to give a specific activity of 793 dpm/mg. After five recrystallizations from methanol, the spe-

TABLE I Recrystallization of biosynthetically labeled sterol fractions with

radwinert sterols

Fraction Authentic Recrystal- Weight Specific lization recovery activity ( d m ) sterol

( w )

Fraction 6 (85,200)'

Fraction 15 (23,800)"

Fraction 6 (4,500)d

cholesterol (23.73)

25-hydroxych0- lesterol (30.00)

25-hydroxycho- lesterol (31.62)

% 0

1 84 2 88 3 80 4 81 5b 71 6 72 0

1 86 2 84 3' 70 4 72 5 62 0

1 86 2 86 3 87 4 79 5 76

dpmlw 3590

3320 3310 3320 3120 3030 3040 793

730 789 866 749 783 133

127 138 134 119 128

a See Fig. 3. After the fifth recrystallization the m.p. was 159-161 "C (m.p.

HPLC on a Resolve column showed identity of peaks of mass

See Fig. 4.4.

(literature) 160-162 "C (8)).

and radioactivity.

FIG. 4. Chromatography of biosynthetically labeled, puta- tive 25-hydroxycholestero1 after recrystallizing it three times with the authentic sterol. The recrystallized sterol (Table I, 1.22 mg, 955 dpm) was chromatographed on a CIS semipreparative column with methanol at a flow rate of 2 ml/min. Aliquots of fractions were assayed for 3H, ( 0 - - -0). The solid line represents Az,o.

cific activity was unchanged. Reverse phase chromatography of the thrice recrystallized sterol showed coincidence of Az10 and radioactivity peaks; all of the radioactivity appeared in a single peak (Fig. 4).

Further Purification by Rechromatography on a CIS Resolve Column-Rechromatography of fractions 6 and 15 from a PPorasil colyann (Fig. 3) on a Resolve CI8 reverse phase column is shown in Fig. 5. Fraction 6 gave coincident peaks of Azlo, radioactivity, and activity in the HMG-CoA reductase

FIG. 5,. Rechromatography of fractions from BPorasil on Cts Re- solve columm. Fractions 15 and 6 from Fig. 3 were rechromatographed on a CIS &solve column (panels A and B, respec- tively), with 8832 methanokwater as the solvent and at a flow rate of 1 ml/min. Aliquots of fractions were assayed for SH (0- - a), for activity in the HMG-CoA reductase repression assay (0. . . .O), and oxysterol protein binding assay (%"X). The solid line represents Azl,,.

r

d \

1 A

I I 1 % R I

2 3 4 S 6 7 8 9 10 I f

I 2 3 4 5 6 7 8 9 1 0 I I FRACTION NO.

repression and binding assays. These peaks coincided with the retention time of standard Z ~ ~ S ) , 2 ~ - e ~ g y c h o ~ e s ~ r o l (Fig. 5, panel 3). Similarly, fraction 15 from the PPorasil column gave major, coincident peaks of mass, ~ a ~ o a c t i ~ t y ~ and bio- iogical activity which co-chromatographed with standard 25- hydroxycholesterol (Fig. 5, panel A) . In addition, a smaller peak of activity detected by the two b i o l o ~ c ~ assays followed the major band. However, this peak was not associated with corresponding radioactivity or Azlo values.

To confirm the identity of the radioactive band from frac-

tion 15 as 25-hydroxycholesterol~ this band was separated, the solvents were evaporated, the residue was dissolved in 1 ml of methanof, 3H was assayed, and 31.62 mg of pure 25-hydroxy- cholesterol was added to give a specific activity of 133 dpm/ mg. The specific activity was not significantly changed after five recrystallizations from methanol (Table I). Gas chroma- tography of an aliquot (radioinert) showed a single peak with the retention time of 25-hydroxycholesterol (data not shown).

Estimations of Cellular Levels of 2413~,25-E~ox~cholesterol and 2 5 - ~ ~ ~ ~ ~ c ~ o ~ s ~ e r o l - ~ s t i m a t e s by several methods of

14578 Regulatory Uxysterols in Cultured Celk

TABLE I1 Concentrations of 24fS),25-epoxychoEesteroE and 25-

hydroxycholesterol in Dede cell cultures Values are based upon me~urements shown in Fig. 4 after correc-

tion for recovery, which was 56% for the epoxide and 44% for the diol.

EpowchoIesterol 25-HydroxycholesteroI 24(5),25-

Measurement Prep- Prep- Prep- Prep- Prep-

tion tion tion tion tion ara- ara- ara- ara- ara-

1 2 3 1 2 nglng cellular protein

A210 19 76 50 8.6 16.4 Repression of HMG-CoA 58 53 9.1

Binding assay 62 77 51 5.5 11.1 Gas chromatoprauhv

reductase

Prep-

tion ara-

3

13.3 14.4

7.2 13.7

Average -C S.E "

56 k 6 11.0 2 1.2

TABLE I11 Uptake of 25-hyd~xycholesterol and 24fS)25-epoxycholesterol by

Dede cells Values represent the average of duplicate determinations.

Sterol Concentra- Incuba-

in medium time

p M n$l min total protem fglceR "/.of Pg/s.

25-Hydroxy- 0.16 44 30 20 23 3.0 cholesterol 120 34 35 4.6

cholesterol 120 46 286 36.9

tion tion Uptake

~ ~ ( S ) , ~ ~ - E P O X Y - 0.89 15 30 26 170 22.0

the concentration of 25-hydroxycholesterol and 24(S),25- epoxycholesterol/milligram of cellular protein are compared in Table 11. For unknown reasons, values for the concentra- tion of 25-hydroxycholesterol as determined by the binding assay are somewhat lower than those obtained by the other methods. Possibly some minor contaminant interfered selec- tively with the binding assay. It is also possible that the apparent discrepancy between the reductase repression and binding assay is largely due to accumulated error. A previous estimate of standard error in replicate determinations with the reductase repression assay was e l2% and with the binding assay, rt7% (1). Protein ConcentrationfDede cell was deter- mined to be 0.13 ng and, taking the average of the values for the sterol concentrations given in Table 11, the concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholestero1 are calculated to be 7.2 and 1.5 fg/cell.

In order to determine whether or not the intracellular concentrations found for the two oxysterols are within the ranges that are required for the regulation of HMG-CoA reductase, we determined the intracellular accumulation of exogenous 3H-oxysterols under conditions wherein they caused 50% repression of the reductase (i.e. the diol and epoxide were present in the culture medium at concentrations of 0.16 and 0.89 p ~ , respectively). As shown in Table 111 the amounts of the two sterols taken up per cell at 30 min were 2-3 times the endogenous amounts found in untreated Dede cells.

DISCUSSION

Two oxysterols, 24(S),25-epoxycholesterol and 25-hydrox- ycholesterol, accounted for the vast majority of the HMG- CoA reductase repression activity in the cellular sterol frac- tion. The presence in Dede cells of 24(S),25-epoxycholesterol in a concentration that appears to be within the range required

for the regulation of HMG-CoA reductase is consistent with levels previously found in human livers2 Thus, this sterol could be involved in the regulation of cholesterol synthesis in a variety of tissues and cells. A regulatory role for the epoxide is attractive because its origination via a branch in the sterol biosynthesis pathway at the level of squalene 2,3(S)-oxide suggests possible mechanisms by which its concentration might be varied. Studies with inhibitors of oxidosqualene cyclase indicate that squalene 2,3(S);22(S),23-dioxide accu- mulates when cyclase activity is inhibited (11, 12). Thus, the level of the activity of squalene epoxidase or of oxidosqualene cyclase could regulate the concentration of 24(S),25-epoxy- cholesterol. It has been reported that the cyclization of squa- lene is one of several steps in the pathway that is depressed in the livers of rats fed cholesterol (26), and in human fibro- blasts administered low density lipoprotein (22). However, the evidence for this, based upon the inco~oration of [3H] mevalonate into various cholesterol precursors, is indirect and inconclusive.

Although the concentration of 25-hydroxycholesterol found in the cells was lower than that of the epoxide, its potency is higher so that the levels of repressor activities attributable to the two sterols were similar. The metabolic origin of the cellular 25-hydroxycholesterol is not known at present. Pre- vious studies indicate that 24(S),25-epoxycholesterol is not a metabolic precursor of 25-hydroxycholesterol in liver homog- enates or in cultured fibroblasts (9): Hydroxylation at C-25 of cholesterol is catalyzed by a hepatic mi~chondrial cyto- chrome P-450 (6). However, metabolic production of the 3@,25-diol from cholesterol in other tissues or cells has, to our knowledge, not been demonstrated. The possibility that 25- hydroxycholesterol may have arisen by auto-oxidation of cho- lesterol or desmosterol during the extraction and fractionation of the sterols is a concern. Extensive precautions, including addition of antioxidants at all stages, rapid processing and cold storage, were taken to prevent auto-oxidation reactions. The fact that control experiments with a mixture of the principal sterols present in the cells did not result in the production of either 24(S),25-epoxycholesterol or 25-hydrox- ycholesterol, indicates that the precautions were sufficient to prevent detectable auto-oxidation. Further evidence against auto-oxidation as the source of either the epoxide or the diol was the absence of any detectable level of 7-ketocholesterol or 7@-hydroxycholesterol, which are major products of choles- terol auto-oxidation (23). Although enzymatic reactions lead- ing to 25-hydroxycholesterol in fibroblasts have not yet been identified, preliminary evidence4 that 25-hydroxycholesterol and 24(S),25-epoxycholesterol are converted to more polar products by cultured fibroblasts, suggests that a system or systems for degrading and inactivating the sterols may be present.

Only a relatively small amount of activity in the HMG- CoA repression assay which was not attributable to 24(S),25- epoxycholesterol or to 25-hydroxycholesterol was detected in the cellular sterol fraction. A small amount of activity asso- ciated with what is thought to be a derivative of 7-dehydro- cholesterol (fraction 10, Fig. 3), may have arisen by photolysis of 7-dehydrocholesterol during extraction and fractionation or by photolysis of cholesta-5,7,24-trien-3@-ol during the assay for repression of HMG-CoA reductase. A very low level of repressor activity found in an early region of the pPorasil chromatogram could be due in part to a small amount (C4 ng/pg protein) of 24(S),25-oxidolanosterol. However, the

F. R. Taylor, A. A. Kandutsch, and T. A. Spencer, unpublished data.

Regulatory Oxysterols in Cultured Cells 14579

source of this minor activity has not been conclusively iden- tified.

The concentrations of 24(S),25-epoxycholesterol(56 ng/mg protein, 7.2 fg/cell) and 25-hydroxycholesterol (11 ng/mg protein, 1.5 fg/cell) found in the cultures appear to be in the range required for repression of HMG-CoA reductase, since these amounts are about one-half to one-third of the concen- trations of the corresponding, exogenously-added, radioactive sterols that accumulate in the cells under conditions wherein HMG-CoA reductase is repressed. Comparison of the endog- enous levels of the epoxide and diol with the intracellular concentrations of tritiated sterols taken up from the culture medium over a 30-min incubation period was taken as inform- ative because we have shown previously that binding of ex- ogenous 25-hydroxycholesterol to the cytosolic oxysterol binding protein in L cell cultures reaches a steady state equilibrium by 30 min (28). Thus the maximum repressor activity may be attained by this time. These measurements of oxysterol uptake per cell do not, however, allow calculation of total intracellular concentrations because efflux of unla- beled endogenous oxysterols may have occurred during the incubation period (28). The concentration of cholesterol in the cells as determined by gas-chromatography was 22 2 3 rg/mg cellular protein or 2840 fg/cell. Thus the concentration of 24(S),25-epoxycholesterol in the Dede cell cultures was 0.25% of that of cholesterol, similar to the ratio found in human liver.'

Although these results suggest that 24(S),25-epoxycholes- terol and/or 25-hydroxycholesterol could be involved in the regulation of HMG-CoA reductase, direct evidence correlating changes in HMG-CoA reductase with those in the concentra- tion of either sterol is still lacking. Clear experiments to establish such a correlation are not obvious since the only agents that effectively modulate the synthesis of HMG-CoA reductase in cell cultures are oxysterols, lipoproteins, which contain oxysterols, (29, 301, and high concentrations of mev- alonic acid.4 Changes in oxysterol concentrations in the pres- ence of high concentrations of mevalonic acid may not be physiologically relevant, since an increased flow of sterol precursors causes increased accumulation of a number of sterol intermediates. It may be useful to look for correlation in cell cultures at different stages of confluency because HMG-CoA reductase activity declines with increasing cell density. However, this decline may be due to less specific regulatory mechanisms that affect levels of many proteins. It is possible that more definitive tests of the hypothesis that 24(S),25-epoxycholesterol and/or 25-hydroxycholesterol are physiological regulators of HMG-CoA reductase can be made by determining levels of the oxysterols in organs and tissues with varying levels of the reductase in vivo.

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20. 21. 22. 23.

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