Altered regulation of cholesterol and cholesteryl ester synthesis in Chinese-hamster ovary cells overexpressing the oxysterol-binding protein is dependent on the pleckstrin homology domain.
Oxysterol-binding protein (OSBP) is a high-affinity receptor for a variety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To examine a potential role for OSBP in regulating cholesterol metabolism, we stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines overexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in cholesteryl ester synthesis when cultured in medium with delipidated serum, 25-hydroxycholesterol or low-density lipoprotein (LDL). CHO-OSBP cells showed a 40-60% decrease in acyl-CoA:cholesterol acyltransferase activity and mRNA, a 50% elevation in mRNA for three sterol-regulated genes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [14C]acetate incorporation into cholesterol. CHO-K1 cells overexpressing two OSBP mutants with a complete or N-terminal deletion of the pleckstrin homology (PH) domain had cholesterol esterification and synthesis rates that were similar to those shown by mock-transfected controls. Unlike wild-type OSBP, both PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presence of 25-hydroxycholesterol. CHO-K1 cells overexpressing OSBP have pronounced alterations in cholesterol esterification and synthesis, indicating a potential role for this receptor in cholesterol homoeostasis. The phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localization of OSBP to the Golgi apparatus.
Lagace
T A
TA
Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia, Canada.
Byers
D M
DM
Cook
H W
HW
Ridgway
N D
ND
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry / Q000502:physiology*; D016466:CHO Cells; D002784:Cholesterol / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002788:Cholesterol Esters / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004951:Esterification; D005786:Gene Expression Regulation / Q000187:drug effects; D006888:Hydroxycholesterols / Q000494:pharmacology; D010750:Phosphoproteins; D012333:RNA, Messenger / Q000378:metabolism; D011987:Receptors, Steroid / Q000096:biosynthesis* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D002785:Sterol O-Acyltransferase / Q000187:drug effects / Q000235:genetics; D000098825:Oxysterol Binding Proteins
D001798:Blood Proteins; D002788:Cholesterol Esters; D006888:Hydroxycholesterols; D010750:Phosphoproteins; D012333:RNA, Messenger; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; C039077:platelet protein P47; C007997:25-hydroxycholesterol; D002784:Cholesterol; D002785:Sterol O-Acyltransferase