Chinese hamster ovary cells overexpressing the oxysterol binding protein (OSBP) display enhanced synthesis of sphingomyelin in response to 25- hydroxycholesterol

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.