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 var iety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To exa mine a potential role for OSBP in regulating cholesterol metabolism, w e stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines ove rexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in chol esteryl ester synthesis when cultured in medium with delipidated serum , 25-hydroxycholesterol or low-density lipoprotein (LDL). CHO-OSBP cel ls showed a 40-60% decrease in acyl-CoA:cholesterol acyltransferase ac tivity and mRNA, a 50% elevation in mRNA for three sterol-regulated ge nes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [C-14]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, bo th PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presenc e of 25-hydroxycholesterol, CHO-K1 cells overexpressing OSBP have pron ounced alterations in cholesterol esterification and synthesis, indica ting a potential role for this receptor in cholesterol homoeostasis. T he phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localiz ation of OSBP to the Golgi apparatus.