The Hypocholesterolemic Agent LY295427 Reverses Suppression of Sterol Regulatory Element-binding Protein Processing Mediated by Oxysterols

Bethany A Janowski,Bei Shan,David W Russell

doi:10.1074/jbc.m108348200

Abstract

The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4-6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action.

Highlights

The accumulation of cholesterol1 in cells activates a feedback mechanism that delimits further buildup of this essential but toxic lipid [1]
Micromolar concentrations of LY295427 induced the metabolism of low density lipoprotein (LDL) in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other sterol regulatory element-binding protein (SREBP) target genes
Cells grown in lipoprotein-deficient medium, conditions that result in the induction of LDL receptor expression, metabolized microgram amounts of LDL

Summary

EXPERIMENTAL PROCEDURES

Materials—LY295427 and LY306039 were kind gifts of Julio Medina (Tularik Inc., South San Francisco, CA), and Robert A. SRD-12B and SRD-13A are CHO-7-derived lines that are deficient in SCAP [23] and the Site 1 protease [24], respectively Both SRD cell types were seeded at a density of 5 ϫ 105 cells/ 10-cm plate on day 0 of an experiment and maintained in medium B (1:1 mixture of Ham’s F-12 medium and DMEM containing 5% (v/v) fetal calf serum (FCS), 100 units/ml penicillin, 100 ␮g/ml streptomycin sulfate, 5 ␮g/ml cholesterol, 20 ␮M oleate, and 1 mM sodium mevalonate) at 37 °C and 8 –9% CO2. On day 5, the medium was removed, the cells were washed once with PBS, and fresh DMEM medium containing 10% (v/v) HLPPS supplemented with 30 ␮M LY295427, 0.4 ␮g/ml 25-hydroxycholesterol plus 5 ␮g/ml cholesterol (Sterols), 10 ␮g/ml LDL, or 10% (v/v) FCS as indicated, were added to the dishes. On day 7, the medium was removed, the cells were washed once with PBS, and DMEM containing 2 mg/ml BSA, the supplements described above, and 10 ␮g/ml [125I]LDL were added as indicated. The numbers shown (means of triplicate determinations) represent high affinity values, which were calculated by subtracting the values obtained in the presence of excess unlabeled LDL (500 ␮g/ml) from those obtained in the absence of unlabeled LDL

Experiment B

RESULTS

Farnesyl diphosphate synthase

DISCUSSION