We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).
Anilides/*pharmacology ; Bile/metabolism ; Cells, Cultured ; Cholesterol/*metabolism ; Cholesterol Esters/metabolism ; DNA-Binding Proteins/agonists/*metabolism ; Enzyme Inhibitors/pharmacology ; Gene Expression Regulation, Enzymologic/drug effects ; Hepatocytes/*drug effects/metabolism ; Humans ; Lipid Metabolism/drug effects/genetics ; Macrophages/*drug effects/metabolism ; Models, Biological ; Oleic Acids/*pharmacology ; Receptors, Cytoplasmic and Nuclear/agonists/*metabolism ; Sterol O-Acyltransferase/*antagonists & inhibitors/physiology ; Transcription Factors/agonists/*metabolism

Several new drug targets of anti-atherosclerosis, emerging in the recent years, such as PPAR agonists, cholesteryl ester transfer protein (CETP) inhibitors, infusion of apolipoprotein A-I (apoA-I), liver X receptor (LXR) activators and phospholipid transfer protein (PLTP) inhibitors etc were reviewed.
Apolipoprotein A-I ; therapeutic use ; Atherosclerosis ; drug therapy ; metabolism ; Benzoates ; chemistry ; therapeutic use ; Benzylamines ; chemistry ; therapeutic use ; Cholesterol Ester Transfer Proteins ; antagonists & inhibitors ; metabolism ; DNA-Binding Proteins ; agonists ; metabolism ; Humans ; Liver X Receptors ; Molecular Structure ; Orphan Nuclear Receptors ; Oxazines ; chemistry ; therapeutic use ; Peroxisome Proliferator-Activated Receptors ; agonists ; metabolism ; Phenylpropionates ; chemistry ; therapeutic use ; Quinolines ; chemistry ; therapeutic use ; Receptors, Cytoplasmic and Nuclear ; agonists ; metabolism

Estrogen Receptor (ERalpha) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERalpha for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERalpha modulator. We cloned the ERalpha LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Eralpha(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERalpha(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERalpha modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 micromol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 micromol/L. Using this screening model, we discovered four ERalpha agonists from 2000 natural and synthetic compounds.
3T3-L1 Cells ; Animals ; Chimera ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; Estrogen Receptor Modulators ; chemistry ; isolation & purification ; Estrogen Receptor alpha ; agonists ; Genes, Reporter ; genetics ; Genistein ; chemistry ; isolation & purification ; HeLa Cells ; Humans ; Luciferases ; genetics ; metabolism ; Mice ; Models, Chemical ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Transfection