Abstract
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.
Highlights
From $The Jackson Laboratory, Bar Harbor, Maine 04609 and the §Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755
The two new sterols the level of HMG-CoA reductase do not involve addition of are believed to be 32-oxolanosterol and 32-hydroxy- oxysterols to, or subtraction of oxysterols from, the culture. lanosterol on the basis of high performance liquid chroO- ne such method is to repress the reductase by adding a high matography (HPLC) retention times and mass spectrcoo-ncentration of mevalonate to the culture medium [6,7,8]
A metric and nuclear magnetic resonance spectroscopic postulated explanation for this effect of mevalonate is that data andby NaBH4reduction of the putative aldehyde the rate-limiting step in the pathway is by-passed by high to materialwhich had the HPLC retention time of the levels of this committed precursor, causing the accumulation putative alcohol
Summary
285"Con a 6-foot column of 4% OV 101 on Anakrom Q The cultures were incubated for 5 hat 37 "C,and the plate was upon the basis of our previous work (l),sterols present in placed in liquiNd, forstorage.To assay HMG-CoA reductase activity, fractions 4 and 11of Fig. 2 cochromatographed with 24(S),25-. Ethanol (0.25ml) and toluene-based scintillation fluid (5 ml) were added, and the mixture was assayed for 3Hand 14C.Concentraverse-phase columnof the two unidentified active stewroelrse similar to each other awnedre considerably shorter than those of the two oxygenated lanostenol standards (C and D). authentic 32-oxolanosterol and 32-hydroxylanosterol were not available for comparison with the cellular sterols tions of HPLC fractions, purified cellular sterols, or oxysterol stan- (but see below), thechromatographicbehavior of thetwo dards required to repress the reductase by 50% were estimated from plots oflog concentration against reductaseactivity as described previously [1]. Undetrhe conditions of unknown sterols on pPorasicl learly distinguished them from the assay, 0.16 pM (6.6 ng) 25-hydroxycholestero was sufficient to 24(S),25-oxidolanosterol,which had retention times of 8.2 repress the reductase by 50%
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