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.
Lagace
T A
TA
Atlantic Research Centre, and the Departments of Pediatrics and Biochemistry, Dalhousie University, 5849 University Avenue, Halifax, Nova Scotia, Canada B3H 4H7.
Byers
D M
DM
Cook
H W
HW
Ridgway
N D
ND
D000818:Animals; D016466:CHO Cells; D002518:Ceramides / Q000378:metabolism; D002784:Cholesterol / Q000096:biosynthesis; D002788:Cholesterol Esters / Q000096:biosynthesis; D006224:Cricetinae; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007700:Kinetics; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism*; D013109:Sphingomyelins / Q000096:biosynthesis*; D014162:Transfection; D000098825:Oxysterol Binding Proteins
D002518:Ceramides; D002788:Cholesterol Esters; D006888:Hydroxycholesterols; D011987:Receptors, Steroid; D013109:Sphingomyelins; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol; D002784:Cholesterol