Abstract
The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression.
Highlights
The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule
We show in this study that forkhead transcription factor 4 (FoxO4) interacts with the mature nuclear form of sterol regulatory element binding protein2 and hypoxia inducible factor (HIF)2␣, singly or in combination, to regulate lanosterol 14␣ demethylase (CYP51) expression
thin-layer chromatography (TLC) of lipid extracts from 14C-acetate-exposed cells revealed in hypoxia a near equal distribution of the radioactive label between cholesterol and a major lipid species that migrated similar to the lanosterol and DHL markers (Fig. 2A, B)
Summary
The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction that occurs at the top of this distant pathway is the demethylation of lanosterol at C14 by sterol 14␣ demethylase, known as CYP51 [1] This reaction occurs either directly after lanosterol biosynthesis or following its reduction to dihydrolanosterol (DHL) by sterol 24-reductase (DHCR24) in the Bloch or KandutschRussell arms of the distal pathway, respectively (Fig. 1). Referred to as the early steps of cholesterol biosynthesis, requires a single oxygen molecule for the conversion of squalene to lanosterol, the distal pathway consumes 10 additional oxygen molecules, three of which are required for the lanosterol 14␣ demethylation step This oxidative step is most sensitive to reduced oxygen availability and is likely to be coupled in hypoxic states with regulatory mechanisms that conserve oxygen to ensure cell survival.
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