261

Calmodulin Antagonists Suppress Cholesterol Synthesis by Inhibiting Sterol h 24 R e d u c t a s e Ivan Filipovic* and |ckhar t Buddecke Institute of Physiological Chemistry, University of Muenster, Waldeyerstrasse 15, D-44OO Muenster, Federal Republic of Germany

Preincubation of hepatoma cells and human skin fibro- blasts in the presence of the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chlor(~l-naphtha- lene sulfonamide resulted in a dose-dependent suppres- sion of [14C]mevalonolactone incorporation into choles- terol. At a calmodulin antagonist concentration of 25 ~mol, the incorporation of [~4C]mevalonolactone into cel- lular cholesterol was suppressed to about 30% (hepatoma cells) and 10% (human skin fibroblasts) of control values. When the total nonsaponifiable [~4C]lipids were separated and analyzed by two-dimensional thin layer chromatog- raphy, an accumulation of [~4C]desmosterol was observed along with reduced formation of [~4C]cholesterol. How- ever, when cells were preincuhated in the presence of [~4C]dihydrolanosterol, [~4C]cholesterol formation was not inhibited by the calmodulin antagonists. About 25% of the cell-associated dihydrolanosterol radioactivity was converted to cholesterol in both control and calmodulin antagonist-pretreated cells. The data suggest that cal- modulin antagonists prevent the conversion of desmo- sterol into cholesterol by inhibiting sterol h 24 reductase and that the enzymes catalyzing sterol ring modifications are not affected by the inhibitors. Lipids 22, 261-265 (1987).

Inhibitors of cholesterol biosynthesis are valuable tools for studying the regulation of cholesterol metabolism in cultured ceils. Compactin (1), 3/3- [2-(diethylamino)ethoxyl]- androst-5-en-17-one hydrochloride (U18666 A) (2,3), 4, 4,10[~-trimethyl-trans-decal-3[~-ol (TMD)(4) and 1-[p-(f~- diethylaminoethoxy)-phenyl]-l-(p-tolyl)-2-(p-chlorophenyl)- ethanol (MER 29, Triparanol) (5) have been found to in- hibit cholesterol biosynthesis by acting at the level of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA; EC 1.1.1.34) reductase (1-3), 2,3-oxidosqualene cyclase (EC 5.4.99.7) (2,4) and sterol h 24 reductase (desmosterol reduc- tase, EC 1.3.1.?) (5), respectively. The results obtained with these inhibitors suggest that oxysterols derived from squalene 2,3:22,23 dioxide may also act as physiological regulators of HMG CoA reductase (3).

Recently, the specific calmodulin antagonists N-(6- aminohexyl)-5-chloro-l-naphthalene sulfonamide (W-7) and trifluoperazine have been shown to suppress the cho- lesterol synthesis in human skin fibroblasts. A concomi- tant stimulation of low density lipoprotein receptor syn- thesis was found to be an independent effect (6). The pres- ent investigation describes the inhibitory effect of the calmodulln antagonists W-7 and trifluoperazine on the cholesterol synthesis in hepatoma cells and human skin fibroblasts. An analysis of the intermediates of choles- terol synthesis accumulated under the influence of cal- modulln antagonists gives evidence for a preferential in- hibition of the conversion of desmosterol to cholesterol by blocking sterol h ~4 reductase.

*To whom correspondence should be addressed.

MATERIALS AND METHODS

W-7 was obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Trifluoperazine was purchased from Sigma (Tauf- kitchen, Federal Republic of Germany). 25-Hydroxycho- lesterol was obtained from Steraloids Inc. (Wilton, New Hampshire). Lanosterol, cholesterol, squalene and desmo- sterol were products of Sigma. Lanosterol was purified from contaminating dihydrolanosterol according to ref. 8. [2-x4C]Mevalonic acid lactone and [26,27JH]25-hydroxy- cholesterol were purchased from NEN (Dreieich, Federal Republic of Germany). All other chemicals and organic solvents were from reputable sources and of analytical grade.

Squalene-2,3-oxide and squalene-2,3:22,23-dioxide were synthesized according to Chang et al. (4) and Field and Holmlund (7). ['4C]Dihydrolanosterol was synthesized en- zymatically from ['4C]mevalonolactone according to the procedure described by Gibbons and Mitropoulos (8). Products of synthesis were acetylated and purified by thin layer chromatography (TLC) on silver nitrate-im- pregnated silica gel plates and visualized by radiofluorog- raphy. Acetyldihydrolanosterol was saponified, and the resulting dihydrolanosterol was extracted with hexane and purified by TLC. [~H]Desmosterol was synthesized from [3H]25-hydroxycholesterol according to the method of Svoboda and Thompson (9).

Cells. Human fibroblasts from skin explants of healthy donors were cultured as described elsewhere (6). Hepa- toma cell line HepG2 was a gift of A. Schwarz, Depart- ment of Pediatrics, Harvard University (Boston, Massa- chusetts). Cells were grown in minimal essential medium supplemented with essential amino acids and 10-20% fetal calf serum. All experiments were performed with cells grown to confluency (2.5-3.0 X 106 cells/25 cm 2 flask).

Incubation conditions. Cells were preincubated for 24 hr in lipoprotein-deficient medium and 12 hr in the pres- ence of the calmodulin antagonists prior to the addition of the radioactive metabolic precursors [~4C]mevalono- lactone or [14C]dihydrolanosterol in absolute alcohol (0.1% by vol). The incubation was stopped by extensive wash- ing of cell layers with ice-cold buffered saline. Ceils were scraped from the culture vessels with a rubber policeman and harvested by centrifugation.

Total lipids were obtained by three cycles of vortexing the cells in chloroform/methanol (2:1, v/v), extracted for 8 hr and saponified at 80 C for 30 rain with 1 M KOH in methanol/benzene (4:1, v/v). Water was added, and the nonsaponifiable lipids were extracted with hexane. The solvent was evaporated using a stream of N2. Lipids were solubflized in small volumes of chloroform/methanol (2:1, v/v) and submitted to two-dimensional TLC as described by Sexton et al. (2). Radioactive lipid (-containing) spots were visualized by radiofluorography, scraped into vials containing scintillation fluid and counted for radioactiv- ity. When individual lipid fractions were further analyzed by TLC, the radioactive spots were scraped into vials

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I. FILIPOVIC AND E. BUDDECKE

containing chloroform/methanol (2:1, v/v) and extracted. Synthetic products were tentatively identified by cochro- matography using known nonradioactive reference sub- stances. The positions of the lipids were ascertained by radiofluorography followed by spraying the plates with concentrated sulfuric acid and incubation at 120 C. To establish unequivocally the identity of individual sterol fractions, the radioactive lipids were acetylated 18) and cochromatographed together with correspondingly acety- lated reference substances on 10% silver nitrate-impreg- nated silica gel plates in hexane/benzene (50:50, v/v).

In vitro assay of sterol h 24 reductase. Microsomes and postmicrosomal fractions were prepared from rat liver homogenates according to the method described by Dempsey (10). Microsomal pellets were washed twice with 0.1 M potassium phosphate buffer, pH 7.3, and recen- trifuged for 30 min at 105,000 • g. Incubation mixtures contained 1 ml of microsomes (about 10 mg protein} suspended in 0.1 M potassium phosphate buffer, pH 8.3, 2.5 ml (18 mg/ml) of 105,000 X g supernatant and 85 X 103 cpm (1.5 ~g) of [3H]desmosterol in 150 ~1 of propylene glycol. Following the addition of 1 ~l/ml of 25 mM trifluoperazine or W-7 in dimethylsulfoxide (1 ~l/ml), the mixture was incubated for 15 min at 37 C. The enzymatic reaction was started by adding NADPH in a small volume of incubation buffer to a final concentration of 0.45 mM. The incubation was terminated after 2 hr by adding 15 ml chloroform/methanol (2:1, v/v) and extrac- tion of lipids. The extraction procedure was repeated three times. The lipid extracts were pooled and the organic solvents were evaporated under vacuum. The total lipids were acetylated (8) and extracted in hexane, and aliquots were submitted to separation on 10% silver nitrate- impregnated silica gel plates in hexane/benzene (50:50, v/v). Radioactive spots were visualized by radiofluorog- raphy, scraped into scintillation fluid-containing vials and counted for radioactivity.

RESULTS

Calmodulin antagonists W-7 and trifluoperazine preven t conversion of desmosterol to cholesterol. Hepatoma cells incorporated ~4C radioactivity from [2-'4C]mevalonic acid lactone during a 6-hr pulse period preferentially into cholesterol. Intermediates of cholesterol synthesis (lano- sterol, dihydrolanosterol and desmosterol) were detect- able as minor components, which were, however, con- verted to cholesterol during an 18-hr chase period (Fig. 1A).

Preincubation of the cells in 25 ~M W-7 or trifluo- perazine led to an accumulation of [~C]desmosterol {spot no. 6 of Figs. 1B and 1C). In addition, squalene 2,3-oxide and 2,3:22,23 squalene dioxide, an oxygenated by-product of sterol synthesis, accumulated within nonsaponifiable lipids. In a pulse-chase experiment, W-7 and trifluo- perazine partially or almost completely prevented the con- version of desmosterol to cholesterol {Figs. 1B and 1C).

Desmosterol and cholesterol {spot numbers 6 and 5 of Fig. 1) were identified as acetyl derivatives. To do this, spots 6 and 5 were recovered from TLC plates, acetylated with acetic anhydride and cocrystallized with authentic acetyl derivatives of cholesterol and desmosterol in various solvent systems. In all systems, radioactive, acetylated material from the spots cochromatographed

FIG. I. Effect of N-(f~aminohexyl)-5-chloro-l-naphthalene sulfon- amide (W-7) and trifluoperazine on sterol synthesis in HepG2 cells. Cells were preineubated for 12 hr in the presence of 25 ~M W-7 or trifluoperazine prior to addition of 5 ~Ci/ml of [2-14C]mevalonolac - tone, and the incubation continued for 6 hr. In pulse-chase ex- periments the incubation in the presence of the radioactivity was terminated after 3 hr by discarding the radioactive medium. Cell layers were washed three t imes with fresh nonradioactive medium and then maintained for 18 hr either in the presence or absence of the indicated concentrations of the calmodulin antagonists. Total lipids of harvested cells were extracted with chloroform/methanol {2:1, v/v) several t imes and processed as described in Materials and Methods. The nonsaponifiable lipids were submitted to separation on silica gel plates by two-dimensionai thin layer chroamtography as described in Materials and Methods. Radioactive spots were identified by radiofluorography. Identified lipids are as follows: I, squalene-2,3-oxide; 2, squalene-2,3:22,23-dioxide; 3, dihydrolano- sterol; 4, lanosterol; 5, cholesterol; 6, desmosterol.

with reference acetyl derivatives. Figure 2 shows fluoro- grams of [~4C]acetylcholesterol and [~4C]acetyldesmo- sterol, which were found in positions virtually identical to those of the nonradioactive reference substances made visible with concentrated sulfphuric acid (not shown}.

Quantitative data on the effect of W-7 and trifluo- perazine on cholesterol synthesis in hepatoma cells and skin fibroblasts are given in Table 1. While the synthesis of total nonsaponifiable lipids in hepatoma cells was sup- pressed by calmodulin antagonists to only 75-80% of control values, the ratio of cholesterol to desmosterol de- creased from 29.6 (control) to 0.54 (W-7) and 0.33 Itrifluoperazine) under the influence of these agents. As demonstrated in Figure 3, the inhibition of cholesterol synthesis by trifluoperazine is dose-dependent.

Analogous results were obtained for human skin fibro- blasts {Table 1). The 14C radioactivity incorporated into

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CALMODULIN ANTAGONISTS INHIBIT STEROL 5 ~4 REDUCTASE

263

TABLE 1

Effect of the Calmodulin Antagonists W-7 and Trifluoperazine on Cholesterol Synthesis from [~4C]Mevalonolactone in HepG2 Cells and Human Skin Fibroblasts

HepG2 cells: [2-~4C]mevalonolactone incorporation (cpm X 10-3/mg cell protein)

Fibroblasts: [2-'4C]mevalonolactone incorporation (cpm X 10-S/mg cell protein)

Final Total Total concentration nonsaponifiable nonsaponifiable

Additions (~M) lipids Desmosterol Cholesterol lipids Desmosterol Cholesterol

None -- 9.6 0.3 8.9 3.7 -- 3.4 W-7 25 7.8 4.6 2.5 3.4 3.1 0.2 Trifluoperazine 25 7.2 5.1 1.7 3.1 2.7 0.1

W-7, N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide. Cells grown to confluency were preincubated for 24 hr in lipoprotein-deficient medium and 12 hr in the presence of the indicated concentrations of the calmodulin antagonists prior to the addition of 5 pCi/ml (HepG2 cells} and 10 ~Ci/ml (fibroblasts) of [2-~'C]mevalonic acid lactone. Incubation with the radioactive precursor was terminated after 6 hr by removing the medium and by extensive washing of the cell layers with Hank's solution. Fresh lipoprotein-deficient medium was then added, and the cells were scraped off the culture vessels and collected by centrifugation 18 hr later. Extraction, saponification and separa- tion of radioactive sterols were as described in Materials and Methods. Values are means of two experiments made in duplicate.

FIG. 2. Identification of synthetic products accumulated in HepG2 cells during a pulse-chase experiment in the presence of trifluo- perazine. Radioactive spots designated 5 and 6 in Fig. 1 from pulse- chased and trifluoperazine*pretreated cells were scraped off into vials containing chloroform/methanol (2:1, v/v). The extracted lipids were acetylated and chromatographed on silver nitrate-impregnated silica gel G plates (Materials and Methods). Nonradioactive acetylcholes- terol and acetyldesmosterol were cochromatographed. The positions of lipids on developed plates were determined by radiofluorography and by spraying the plates with concentrated sulphuric acid.

the total nonsaponifiable lipids of fibroblasts was two to three times less than that incorporated into the lipids of hepatoma cells. Furthermore, the shift in the ratio of ch~ lesterol to desmosterol under the influence of calmodulin

~ O ~ O

-8~ ~'2

o

100

75

50

25-

i

lO 3b 4o

Trifluoperazlne (I~M)

FIG. 3. Inhibition of sterol synthesis in HepG2 cells relative to trifluoperazine concentration. Cells were preincubated for 24 hr in lipoprotein-deficient medium and 12 hr in the presence of the in- dicated concentrations of trifluoperazine. Following incubation for 3 hr with 5 ~Ci/ml [2-14C]mevalonolactone, the lipids were extracted from the cells as described in Materials and Methods and Fig. 1. Lipids were separated by twwdimensional thin layer chromatog- raphy. Total sterol synthesis was estimated by counting an aliquot of nonsaponifiahle lipids.

antagonists was remarkable; the formation of ['4C]choles- terol dropped from 100% {control} to 3-6% (calmodulin antagonists}, which caused desmosterol to accumulate.

Calmodul in a n t a g o n i s t s inh ib i t s tero l M 4 reductase. The conve r s ion of d e s m o s t e r o l to cho les te ro l is c a t a l y z e d b y s t e ro l h ~' r educ t a se . Th i s e n z y m e h a s been r e p o r t e d to be p r e sen t in va r ious cho les te ro l -p roduc ing t i s sues and to r e s ide in t he m i c r o s o m a l f r ac t i on (11), b u t h a s n o t y e t been c h a r a c t e r i z e d in de ta i l . R a t l iver m i c r o s o m e s were u s e d to s t u d y the ef fec t of W-7 and t r i f l uope raz ine on s t e ro l h ~4 r e d u c t a s e a c t i v i t y in v i t ro . The d a t a g iven in Tab le 2 d e m o n s t r a t e t h a t in con t ro l e x p e r i m e n t s a b o u t 60% of t h e [3H]desmos te ro l is c o n v e r t e d w i th in 2 hr to

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I. FILIPOVIC AND E. BUDDECKE

TABLE 2

In Vitro Effect of W-7 and Trifluoperazine on Sterol A 24 Reductase of Isolated Rat Liver Microsomes

Additions ~M

~H radioactivity % of

Desmosterol Cholesterol Substrate (cpm) (cpm) converted

11240 18760 62.5 24150 5980 19.8 25860 5320 17.0

None W-7 25 Trifluoperazine 25

W-7, N-(6-aminohexyl)-5-chlorc~l-naphthalene sulfonamide. ['"C]- Desmosterol (1.5 ~g = 85 • 103 cprrdassay) was used as substrate. Incubation conditions, lipid extraction, separation and acetylation are described in Materials and Methods. Comparable amounts of radioactivity (about 30,000 cpm) of acetylated total [~H]lipids were submitted to thin layer chromatography. All experiments were per- formed in duplicate.

T A B L E 3

Effect of W-7 and Trifluoperazine on Cholesterol Synthesis from [~4C]Dihydrolanosterol in HepG2 Cells

'4C radioactivity Final

concentration Dihydrolanosterol Cholesterol Additions (~M) (cpm : 103) (cpm X 10 ~)

None -- 36.1 9.1 W-7 25 35.3 7.5 Trifluoperazine 25 37.2 6.9

W-7, N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide. Cells were preincubated for 24 hr in lipoprotein-deficient medium and 12 hr in the presence of 25 ~M W-7 or trifluoperazine. Then ['4C]dihydrolan~ sterol (2.5 • l0 s cpm/ml) in absolute alcohol (10 ~l/ml) was added, and incubation was continued for 6 hr. After extensive washing with buffered saline the cells were scraped from the culture vessels and harvested by centrifugation. Lipids were extracted, and aliquots with equal amounts of radioactivity were submitted to separation on paraffin oil-impregnated silica gel G plates using acetone/water satu- rated with paraffin {85:15, v/v) as solvent. Radioactive lipids were detected by fluorography, recovered and counted. The identity of lipid fractions was ascertained by comparison with cochromato- graphed nonradioactive dihydrolanosterol and cholesterol. Values are means of two experiments made in duplicate.

[3H]cholesterol by rat liver microsomes in the presence of NADPH. Under these conditions, W-7 and trifluo- perazine significantly inhibited the enzymatic reduction of desmosterol to cholesterol {30% of control values}.

Enzymatic conversion of dihydrolanosterol to choles- terol is not inhibited by calmodulin antagonists. The en- zymatic conversion of lanosterol to cholesterol is com- prised of several reactions: reduction of the A ~ double bond, removal of three methyl groups and shift of the nuclear double bond from the h s to the A s position. Calmodulin antagonists inhibit predominantly the reduc- tion of the A 24 double bond, because when cells were in- cubated in the presence of ["C]dihydrolanosterol, nearly equal amounts of '4C radioactivity were incorporated

Lanosterol

(4,4,14a-t r i me thy I - cholesta-8,24-dien-3B-ol )

Dihydrolanosterol z~ 24 side chain (4,4, la~-tr imethyl - i ntermedi ates choles ta-8-en- 3B-ol )

{

saturated side chain intermediates

Cholesterol (cholesta-Sen-3B-ol)

~ A 24 reductase{ i

Desmosterol (cholesta- 5,24-di en- 3B-ol )

reductase

SCHEME 1

into cholesterol in both control and calmodulin antago- nist-pretreated HepG2 cells {Table 3). Dihydrolanosterol has been found to be a sterol intermediate in the biosyn- thesis of cholesterol and to be convertible to cholesterol (12,13}. However, the reaction sequence dihydrolano- sterol --" cholesterol seems to be bypassed in HepG2 cells; only 25% of the internalized ['~C]dihydrolanosterol was converted to cholesterol by these cells.

DISCUSSION

The present s tudy indicates tha t the calmodulin an- tagonis ts W-7 and trifluoperazine suppress the biosyn- thesis of cholesterol by blocking sterol A 24 reductase. The enzymatic reduction of the A 24 double bond is one of the necessary reactions during cholesterol synthesis. Reduc- tion of the bond may occur at any of several points dur- ing t ransformation of lanosterol to cholesterol. Thus, sterol A 2" reductase may catalyze an initial conversion of lanosterol to 24,25-dihydrolanosterol, which has been found to be convertible to cholesterol {13}. An alternative metabolic pa thway leads via a series of a ~4 side chain in- termediates from lanosterol to desmosterol, which is finally converted to cholesterol by sterol A 24 reductase (see Scheme 1). The finding that the calmodulin antagonists used in this s tudy do not inhibit the conversion of ['~C]24,25-dihydrolanosterol to cholesterol (Table 3), but cause an accumulation of desmosterol {Table 1), suggest

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a) tha t calmodulin antagonist- induced inhibition of sterol h 24 reductase is relatively specific and b) tha t the enzymes cata lyzing sterol r ing modifications are not affected by the inhibitors.

Calmodulin an tagonis t s are repor ted to be poten t and specific inhibitors of calmodulin-mediated react ions and calmodulin-dependent enzyme activities (14). Such inhibi- t ion appears to result f rom conformational restr ict ions conferred upon calmodulin by the bond an tagonis t (15). Our results, however, do not allow any conclusion as to whether sterol 524 reductase is a calcium calmodulin- regulated enzyme. In this regard, the conversion of [3H]- desmosterol to [3H]cholesterol by isolated ra t liver micro- somes under in vi tro conditions was found to be inhibited by the calmodulin an tagonis t s (Table 2), bu t the calcium calmodulin concentrat ion of the microsomal fraction was not determined. Furthermore, no information is available to indicate whether the act ivi ty of sterol h 24 reductase is controlled by calcium calmodul in-dependent protein- kinases or by phosphoprote in phosphatases . In view of the previously repor ted inhibition of sterol 624 reductase by U18666 A (2,3). TMD (4) and t r iparanol (16), the en- zyme seems to be highly sensit ive to agents with dis- para te chemical s tructure.

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J. Biol. Chem. 253, 1121-1128.

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4. Chang, T.Y., Schiavoni, E.S. Jr., and McCrae, K.R. (1979) J. BioL Chem. 254, 11258-11263.

5. Steinberg, D., and Avigan, J. {1960} J. Biol. Chem. 235, 3127-3129.

6. Filipovic, I., and Buddecke, E. {1986} Biochim. Biophys. Acta 876, 124-132.

7. Field, R.B., and Holmlund, C.E. {1977} Arch. Biochem. Biophys. 180, 465-471.

8. Gibbons, G.F., and Mitropoulos, K.A. (1972} Biochem. J. 132, 439-448.

9. Svoboda, J.A., and Thompson, M.J. {1967} J. Lipid Res. 8, 152-154.

10. Dempsey, M.E. {1969} Methods Enzymol. 15, 501-514. 11. Steinberg, D., and Avigan, J. (1969} Methods Enzymol. 15,

514-522. 12. Gibbons, G.F., Pullinger, C.R., and Mitropoulos, K.A. (1979)

Biochem. J. 183, 309-15. 13. Frantz, I.D. Jr., and Schroepfer, G.J. Jr. (1967) Ann. Rev.

Biochem. 36, 691-726. 14. Tuana, B.S., and MacLennan, D.H. {1984} J. Biol. Chem. 259,

6979-6983. 15. Dalgarno, D.C., Klevit, R.E., Levine, B.A., Scott, G.M.M.,

Williams, R.J.P., Gergely, J., Zgrabarek, Z., Leavis, P.C., Grand, R.J.A., and Drabikowski, W. {1984} Biochim. Biophys. Acta 791, 164-172.

16. Avigan, J., Goodman, D.S., and Steinberg, D. {1963} J. BioL Chem. 4, 1283-1286.

[Received November 11, 1986]

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