25-Hydroxycholesterol stimulates sphingomyelin synthesis in Chinese hamster ovary cells

In several experimental and pathological conditions the cellular concentrations of cholesterol and sphingomyelin (SM) change coordinately. In an effort to identify factors mediating co-regulation, a class of suppressors of cholesterol synthesis, generically termed oxysterols, were tested for effects on sphingolipid synthesis in Chinese hamster ovary (CHO) cells. 25-Hydroxycholesterol was found to stimulate [3H]serine, [1-3H]palmitate, and [methyl-3H]choline incorporation into sphingomyelin 2- to 3-fold and increase sphingomyelin mass significantly, but did not influence synthesis of other major phospholipids. Maximal labeling of sphingomyelin by [3H]serine was observed 4-6 h after oxysterol addition, and coincided with inhibition of transcription of sterol-regulated genes and activation of cholesteryl ester synthesis. 25-Hydroxycholesterol dose-response curves for activation of sphingomyelin synthesis, suppression of sterol-regulated transcription, and activation of cholesteryl ester synthesis were also similar. Stimulation of SM and glucosylceramide synthesis was observed only with 25-hydroxycholesterol; other oxysterols and cholesterol were ineffective or inhibitory. The effects of 25-hydroxycholesterol on sphingolipid synthesis could not be reproduced by low density lipoprotein (LDL), whole serum, or non-lipoprotein cholesterol in the medium, and stimulation by 25-hydroxycholesterol was evident irrespective of a cholesterol source in the medium. 25-Hydroxycholesterol-treated CHO cells displayed enhanced conversion of [3H]sphinganine-labeled ceramide into sphingomyelin. Sphingomyelin synthesis from N-hexanoyl [3-3H]ceramide and N-hexanoyl [3-3H]dihydroceramide was also increased significantly. Consistent with enhanced ceramide conversion to sphingomyelin, ceramide mass was reduced by 20-40% in 25-hydroxycholesterol-treatment. However, in vitro activity of sphingomyelin synthase (assayed with short-chain ceramide) was not increased in membranes from oxysterol-treated cells. Stimulation of sphingolipid synthesis by 25-hydroxycholesterol is temporally related to effects of this oxysterol on cholesterol metabolism, and is the result of enhanced conversion of ceramide to SM.