Oxygen-Mediated Regulation of Cholesterol Synthesis through Accelerated Degradation of HMG COA Reductase
Nguyen, Andrew Tuan Duc
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Endoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the methylated cholesterol precursors lanosterol and 24,25-dihydrolanosterol. Conversion of these sterols to cholesterol requires the removal of three methyl groups, which consumes nine molecules of oxygen. Here, we report that oxygen deprivation (hypoxia) slows the rate of demethylation of lanosterol and its reduced metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. These methylated sterols serve as one signal to stimulate rapid Insig-mediated degradation of reductase. In addition, hypoxia increases the expression of Insig-2 in a response mediated by hypoxia-inducible factor. Our analysis of the mouse Insig-2 gene revealed the presence of a functional hypoxia response element in the first intron. Importantly, hepatic Insig-2a expression is upregulated in three independent mouse models of hypoxia. These studies establish that Insig-2 is a target gene of hypoxia-inducible factor. The hypoxia-dependent increase in Insig levels confers cells with enhanced sensitivity to sterol-induced degradation of reductase. In this way, hypoxia-inducible factor-mediated induction of Insig-2 provides a second signal for stimulating reductase degradation. To address the specificity of methylated sterols in promoting reductase degradation, we reconstituted Insig-dependent, sterol-accelerated degradation of the membrane domain of mammalian reductase in Drosophila S2 cells. Studies in this system revealed that 24,25-dihydrolanosterol, and lanosterol, is active in accelerating degradation of reductase. These results were confirmed by examining ubiquitination of reductase in vitro using permeabilized mammalian cells. Collectively, these studies show that under hypoxic conditions reductase undergoes accelerated Insig-dependent degradation as the combined result of two events: 1) accumulation of 24,25-dihydrolanosterol and 2) hypoxia-inducible factor-mediated upregulation of Insig-2. Degradation of reductase ultimately slows a rate-determining step in cholesterol synthesis. These results highlight the importance of 24,25-dihydrolanosterol as a physiologic regulator of reductase degradation and define a novel oxygen-sensing mechanism in the mammalian cholesterol biosynthetic pathway.