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

Abstract

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.

Osteoblast-derived Oxysterol Is a Migration-inducing Factor for Human Breast Cancer Cells

“…This result indicates that the effect of OS on the sphingomyelin and cholesterol concentration may be cell-specific. In MCF-7 cells, OS appears to elevate phospholipids and cholesterol with less specificity for sphingomyelin than reported for other cell types (42)(43)(44)(45). The effect of OS on cholesterol was verified by filipin staining of OS-incubated MCF-7 cells, which showed accumulation of cholesterol in lamellipodia or at the membranes of adjacent cells (not shown).…”

“…Binding of OS to ORPs has been shown to participate in the Golgi translocation/anchoring and activation of ORPs (42)(43)(44)(45). It has also been found that an activated, Golgi-localized OS-ORP complex modulates the biosynthesis or transport of sphingomyelin and cholesterol (16 -21, 42-45).…”

The Oxysterol-binding Protein Homologue ORP1L Interacts with Rab7 and Alters Functional Properties of Late Endocytic Compartments

“…The cytosolic oxysterol-binding protein OSBP was identified in the 1980s (Taylor et al, 1984;Taylor and Kandutsch, 1985), and a role for the protein in cholesterol and sphingomyelin metabolism has been implicated previously (Lagace et al, 1997(Lagace et al, , 1999. Families of proteins homologous to OSBP have recently been identified in eukaryotic organisms from yeast to human .…”

Variation in OSBPL10 is associated with dyslipidemia

“…Lagace et al 14 showed that OSBP overexpression in CHO cells results in a 40-60% decrease in acyl-CoA: cholesterol acyltransferase activity and its mRNA, a 50% elevation in the mRNA levels of several sterol regulated genes (LDL receptor, HMG-CoA reductase and HMG-CoA synthase), and an 80% elevation in cholesterol biosynthesis. The observed changes in mRNA levels suggest that OSBP affects transcriptional control of the sterol homeostatic machinery.…”