24(S),25-Epoxycholesterol. Evidence consistent with a role in the regulation of hepatic cholesterogenesis

Previously we showed that 24(S),25-epoxycholesterol is formed from acetate, via squalene 2,3(S),22(S),23-dioxide and 24(S),25-oxidolanosterol, during the normal course of cholesterol biosynthesis in S10 rat liver homogenate (Nelson, J. A., Steckbeck, S. R., and Spencer, T. A. (1981) J. Biol. Chem. 256, 1067-1068; Nelson, J. A., Steckbeck, S. R., and Spencer, T. A. (1981) J. Am. Chem. Soc. 103, 6974-6975). Herein we demonstrate that the nonsaponifiable extract from human liver tissue contains 24(S),25-epoxycholesterol in an amount approximately 10(-3) relative to cholesterol. We show that 24(S),25-epoxycholesterol, like many other oxygenated sterols, represses hydroxymethylglutaryl-CoA reductase activity in cultured cells and binds to the cytosolic oxysterol-binding protein. Furthermore, we show that this epoxide is not rapidly metabolized in cultured cells. These results suggest that 24(S),25-epoxycholesterol may participate in the regulation of hepatic cholesterol metabolism in vivo.     

24,25-Epoxysterol metabolism in cultured mammalian cells and repression of 3-hydroxy-3-methylglutaryl-CoA reductase

In view of the potential importance of 24,25-epoxysterols as intracellular regulators of 3-hydroxy-3-methylglutaryl-CoA reductase, the C-24 epimers of 24,25-oxidolanosterol and 24,25-epoxycholesterol were tested for their biological activity and metabolism in cell cultures. All four compounds produced repression of the reductase in cultured mouse fibroblasts (L cells), and both 24(S)- and 24(R),25-epoxycholesterol exhibited high affinity binding to the cytosolic oxysterol-binding protein. However, binding of the epimeric 24,25-oxidolanosterols was not detected. 24(S),25-Epoxycholesterol was not rapidly metabolized in either L cells or Chinese hamster lung (Dede) cells. 24(S),25-Oxidolanosterol was rapidly converted to 24(S),25-epoxycholesterol in both cell lines. 24(R),25-Oxidolanosterol was converted to 24(R)-hydroxycholesterol in Dede cells, but was converted instead to 24(R),25-epoxycholesterol in L cells, which lack sterol delta 24-reductase activity. Although 24(S),25-oxidolanosterol does not appear to accumulate in these cell cultures, it was found in human liver in about one-fifth the amount of 24(S),25-epoxycholesterol. 24(R),25-Epoxycholesterol was also converted to 24(R)-hydroxycholesterol in Dede cells, but not in L cells. Triparanol inhibited the reduction of the 24(R),25-epoxides in Dede cells, consistent with the idea that this reaction is catalyzed by the delta 24-reductase. 24(R)-Hydroxycholesterol and its 24(S) epimer exhibited affinity for the binding protein and repressed 3-hydroxy-3-methylglutaryl-CoA reductase.     

Photoaffinity labeling of the oxysterol receptor

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.     

Identification of regulatory oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol, in cultured fibroblasts

Biosynthetically tritiated sterols from Chinese hamster lung (Dede) cells were fractionated by high performance liquid chromatography, and fractions were assayed for their ability to repress 3-hydroxy-3-methylglutaryl-CoA reductase in L cell cultures. Most of the activity found was associated with two oxysterols, 24(S),25-epoxycholesterol and 25-hydroxycholesterol. The identities of the two sterols were established by co-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 fg/cell) and 25-hydroxycholesterol (1.5 fg/cell) appear to be within the ranges required for the regulation of HMG-CoA reductase.     

Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures

In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J. Biol. Chem. (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols. The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol. The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate. However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol. These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity.     

Requirement for 24(S),25-epoxycholesterol for the viability of cultured fibroblasts

Previous studies on a somatic cell mutant auxotrophic for mevalonate (Mev-1) have shown that these cells rapidly lose viability when deprived of mevalonic acid in culture medium supplemented with serum cholesterol. Testing of all known end products of mevalonate metabolism in cultured mammalian cells has been conducted to determine the basis for this mevalonate requirement. It has been found that the recently discovered mevalonate metabolite 24(S),25-epoxycholesterol produces a partial restoration of viability of Mev-1 cells starved for mevalonate, whereas other structurally similar oxysterols do not. It appears that 24(S),25-epoxycholesterol has a specific, vital cellular function in CHO-K1 cells.