Abstract
25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells. Concurrent with these effects, 25-hydroxycholesterol also stimulates the synthesis of sphingomyelin in Chinese hamster ovary (CHO)-K1 cells. The role of oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol, in activation of SM synthesis was assessed by overexpression in CHO-K1 cells. When compared to mock transfected controls, three CHO-K1 clones overexpressing OSBP by 10- to 15-fold displayed a 2- to 3-fold enhancement of [3H]serine incorporation into sphingomyelin when treated with 25-hydroxycholesterol. Closer examination of one of these clones (CHO-OSBP cells) revealed a >8.5-fold stimulation of sphingomyelin synthesis after a 6-h treatment with 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-hydroxycholesterol. [3H]serine incorporation into phosphatidylserine, phosphatidylethanolamine, ceramide, or glucosylceramide was affected by <15%. Synthesis of sphingomyelin from exogenous [3H]sphinganine-labeled ceramide was enhanced in overexpressing cells treated with 25-hydroxycholesterol. However, in vitro activities of sphinganine N-acyltransferase, sphingomyelin synthase, and serine palmitoyltransferase were not affected by OSBP overexpression or 25-hydroxycholesterol. Overexpression of OSBP or 25-hydroxycholesterol did not significantly affect the ceramide content of Golgi-enriched fractions from control or overexpressing cells. However, diglyceride mass was reduced in Golgi-enriched fractions from overexpressing cells and by treatment with 25-hydroxycholesterol. Results from overexpressing cells show that OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphingomyelin synthesis. 25-Hydroxycholesterol promotes translocation of OSBP to the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin.
Highlights
25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells
Three clonal Chinese hamster ovary (CHO)-K1 cell lines stably overexpressing rabbit oxysterol binding protein (OSBP) and three cell lines harboring the empty vector were assayed for stimulation of SM synthesis by 25hydroxycholesterol
Our previous finding that 25-hydroxycholesterol stimulated SM synthesis has been extended to show that overexpression of the high affinity oxysterol receptor, OSBP, enhanced this response. 25-Hydroxycholesterol treatment of cell lines overexpressing OSBP resulted in increased SM synthesis relative to controls, but had little effect on [3H]serine incorporation into GluCer, PtdSer, PtdEtn, or ceramide
Summary
25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells. When compared to 25-hydroxycholesterol-treated control cells, [3H]serine incorporation into sphinganine and sphingosine of total sphingolipids of CHO-OSBP cells was increased by 40% When similar experiments were performed in control and overexpressing CHO-K1 cells, 25-hydroxycholesterol stimulated [3H]sphinganine incorporation into SM with a reduction in the level of [3H]ceramide.
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