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

• Published in: Biochemical journal 1997-08, Vol.326 ( Pt 1) (1), p.205-213
• Main Authors: Lagace, T A, Byers, D M, Cook, H W, Ridgway, N D
• Format: Article
• Language: English
• Subjects: Animals; Blood Platelets - chemistry; Blood Proteins - chemistry; Blood Proteins - physiology; CHO Cells; Cholesterol - biosynthesis; Cholesterol - genetics; Cholesterol - metabolism; Cholesterol Esters - biosynthesis; Cholesterol Esters - genetics; Cholesterol Esters - metabolism; Cricetinae; Esterification; Gene Expression Regulation - drug effects; Hydroxycholesterols - pharmacology; Phosphoproteins; Receptors, Steroid - biosynthesis; Receptors, Steroid - physiology; RNA, Messenger - metabolism; Sequence Homology, Amino Acid; Sterol O-Acyltransferase - drug effects; Sterol O-Acyltransferase - genetics
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3260205

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.