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 activa tes cholesterol esterification in a variety of cultured cells, Concurrent w ith these effects, 25-hydroxycholesterol also stimulates the synthesis of s phingomyelin 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 ce lls. When compared to mock transfected controls, three CHO-K1 clones overex pressing OSBP by 10- to 15-fold displayed a 2- to 3-fold enhancement of [H- 3]serine incorporation into sphingomyelin when treated with 25-hydroxychole sterol. Closer examination of one of these clones (CHO-OSBP cells) revealed a >8.5-fold stimulation of sphingomyelin synthesis after a 6-h treatment w ith 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-h ydroxycholesterol. [H-3]serine incorporation into phosphatidylserine, phosp hatidylethanolamine, ceramide, or glucosylceramide was affected by <15%, Sy nthesis of sphingomyelin from exogenous ["H]sphinganine-labeled ceramide wa s enhanced in overexpressing cells treated with 25-hydroxycholesterol, Howe ver, in vitro activities of sphinganine N-acyltransferase, sphingomyelin sy nthase, and serine palmitoyltransferase were not affected by OSBP overexpre ssion or 25-hydroxycholesterol. Overexpression of OSBP or 25-hydroxycholest erol did not significantly affect the ceramide content of Golgi-enriched fr actions from control or overexpressing cells. However, diglyceride mass was reduced in Golgi-enriched fractions from overexpressing cells and by treat ment with 25-hydroxycholesterol. Results from overexpressing cells show tha t OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphi ngomyelin synthesis. 25-Hydroxycholesterol promotes translocation of OSBP t o the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin.