Abstract
Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.
Highlights
Cholesterol homeostasis is governed by its end product, cholesterol, and by oxygenated derivatives of cholesterol, known as oxysterols
We investigated the molecular mechanism through which side chain oxysterols exert their cholesterol homeostatic effects
Of cholesterol homeostatic responses in cells is not mediated by stereospecific oxysterol-protein interactions
Summary
Cell Culture and Chemicals—CHO-K1 cells (American Type Culture Collection) were cultured in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F-12 with 5% (v/v) fetal bovine serum (Sigma), 2 mM glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin. In the SREBP reporter assay, the EC50 for ent25-HC for suppression of sterol-regulated gene expression was 6-fold lower than the EC50 for nat-25-HC, ent-25-HC was still significantly more potent than the steroid ring-modified oxysterols (Fig. 1A). When the assay was performed using a reporter construct driven by a minimal promoter regulated by tandem SRE sequences [15] instead of the human low density lipoprotein receptor promoter, differences between nat- and ent-25-HC were no longer apparent (Fig. 2C), suggesting that oxysterols may modulate expression from the low density lipoprotein receptor promoter through non-SRE sequences and that such a mechanism is likely introducing an enantioselective step into regulation of reporter expression. Using a sterol regulatory element-containing reporter construct as an indicator of the status of SREBP maturation, we examined the ability of both side chain oxysterols (25-HC and 27-HC) and steroid B ring-modified oxysterols (7␣-HC, 7-HC, and 7-KC) to suppress SREBP-dependent gene expression (Fig. 1A). The latter observations suggest that 7-HC and 7-KC exert only a slight condensing or membrane-ordering effect in DOPC monolayers, in contrast to the more substantial nat-25-Hydroxycholesterol ent-25-Hydroxycholesterol condensing effect of cholesterol (Fig. 3F) [18]
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