Synthesis of the oxysterol, 24(S), 25-epoxycholesterol, parallels cholesterol production and may protect against cellular accumulation of newly-synthesized cholesterol

Abstract

Aim: The effects of 24(S),25-epoxycholesterol (24,25EC) on aspects of cholesterol homeostasis is welldocumented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most? Methods:We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14 C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays. Results:We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP transcriptional activity whilst increasing ABCA1 and LXR transcriptional activity. Conclusion:Our results show that 24,25EC synthesis parallels cholesterol synthesis, consistent with this oxysterol functioning as a safety valve to protect against the accumulation of newly-synthesised cholesterol (as opposed to exogenously-derived cholesterol). Considering that 24,25EC is capable of being produced in all cholesterogenic cells, we propose that production of 24,25EC may represent a ubiquitous defence mechanism.

The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii

“…This has been most clearly shown with regard to cholesterol metabolism in which the dioxidosqualene is actually the preferred substrate for lanosterol synthase in the synthesis of the 24,25-epoxycholesterol, which inhibits the reaction with mono-oxidosqualene and serves as part of a feedback control in cholesterol biosynthesis (27,35,36). EPH.…”

Cross-talk between the Androgen Receptor and the Liver X Receptor

“…We tested this hypothesis using the LXRE-luc construct, which has three LXREs in tandem upstream of a viral TK promoter (19). We utilized GW683965 alone because TO901317 antagonizes the AR (31), as observed with our own assays (data not shown).…”

“…Furthermore, because LXRE-luc is driven by both LXR response elements (LXREs) and the viral TK promoter (19), additional cells were transfected with the TK-luc construct instead of LXRE-luc in the same experiment, and relative LXRE-luc activity values were divided by those of TK-luc to determine LXRE-specific promoter activity. For simplicity, this has been depicted as "LXRE-luc activity" in the figures presented here.…”

Antipsychotic drugs upregulate lipogenic gene expression by disrupting intracellular trafficking of lipoprotein-derived cholesterol

“…Liver X receptor promoter activity assay CHO-7 cells were grown in triplicate in 24-well plates, and transfected for 4 h in Medium A (lacking penicillin/streptomycin) with 250 ng 3 Â LXRE luciferase reporter plasmid 21 (or pGL3-basic empty vector) and 25 ng Renilla control plasmid (phRL-TK) using lipofectamine LTX. Cells were then treated for 16 h, washed with PBS and lyzed in passive lysis buffer (Promega).…”

“…Chinese Hamster Ovary (CHO) cells are commonly used in studies on cholesterol metabolism and trafficking. 16,20,21 Earlier cell culture studies showing effects of APDs on SREBP-target gene expression have been conducted in media containing full serum. 4,[6][7][8][9] Here, we used lipoprotein-deficient serum (LPDS) supplemented with the major cholesterol-carrying particle in the bloodstream, LDL, to investigate this phenomenon under controlled conditions, using several approaches to gauge lipoproteinderived cholesterol delivery to the ER.…”