Enhanced Synthesis of the Oxysterol 24( S ),25-Epoxycholesterol in Macrophages by Inhibitors of 2,3-Oxidosqualene:Lanosterol Cyclase

Abstract

Abstract-Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL-induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR-and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis. T he uptake of apolipoprotein (apo) B-containing lipoproteins by macrophages within the arterial wall results in cholesteryl ester (CE) deposition and foam cell formation, a hallmark of early and late atherosclerotic lesions. 1 Foam cell formation is the result of an imbalance in processes mediating lipoprotein uptake and cholesterol efflux. 2 The importance of cholesterol efflux in the maintenance of macrophage cellular cholesterol homeostasis arose from elucidation of mutations in the ATP binding cassette protein (ABC) A1 in Tangier disease, which is characterized by macrophage cholesterol accumulation (reviewed by Attie et al 3 ). ABCA1 is a transmembrane protein that controls the transfer of cholesterol and phospholipids to apoA1, the initial step in HDL formation and reverse cholesterol transport (reviewed by Tall et al, 2 Francis et al, 4 and Oram 5 ). The discovery of ABCA1 and its key role in regulating macrophage cholesterol efflux has stimulated development of therapeutic interventions that enhance the removal of cholesterol from arterial cells as a strategy to reduce atherosclerosis. 2 Expression of...

Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol

“…Previously, we demonstrated that inhibition of OSC by RO0714565 at a dose that maximizes 24(S),25-epoxy synthesis enhanced cholesterol efflux from cultured mouse macrophages, an effect associated with an LXRinduced increase in the expression of ABCA1 and ABCG1. In contrast to the synthetic, nonsteroidal LXR agonist TO-901317, OSC inhibition did not lead to an accumulation of cellular triglycerides, possibly due to the lack of increase in nuclear SREBP-1 (nSREBP-1) (19). However, the underlying mechanism for this effect was not determined.…”

“…Lipids were extracted, saponified, and separated by TLC using Whatman Partisil LK6 TLC plates in hexane/diethyl ether/ acetic acid (60:40:1, v/v/v). A PhosphorImager and ImageQuant software (Amersham Biosciences) were used for quantification of the cholesterol and 24(S),25-epoxy bands (19,23). 24(S),25-epoxy was identified by comparing the R F value with a pure standard (19).…”

“…The synthesis of cholesterol and fatty acid during the 5 h after the preincubation was measured by incorporation of [1-14 C]acetic acid into these lipids. Saponified cellular lipid extracts were separated, and cholesterol was identified by TLC using Whatman Partisil LK6 TLC plates in petroleum ether/diethyl ether/acetic acid (84:15:1, v/v/v) and quantitated as described previously (19). Fatty acids were measured from a second extraction following acidification of the infranatant of the saponified lipid extract as described previously (25).…”

“…LPL activity in the medium of THP-1 cells was determined following a 24-h incubation with OSCi or the indicated LXR agonists (as described above) as the release of free fatty acids from intralipid, an exogenous lipid source (26). Cholesterol efflux was measured using 5 g of cholesterol/ml of acLDL in the presence of OSCi or selected doses of LXR agonists as described previously (19).…”

Identification of human low‐density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

“…There might be species differences between human and rodents. However, Rowe et al [14] reported that T0901317 increased the mRNA expression of LDLR gene in murine macrophage J774A.1 cells. Masson et al [15] reported the similar induction in the liver from C57BL6 mice by T0901317.…”