Browsing by Subject "Hypoxia-Inducible Factor 1, alpha Subunit"
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Item Oxygen-Mediated Regulation of Cholesterol Synthesis Through Accelerated Degradation of HMG COA Reductase(2009-09-04) Nguyen, Andrew Tuan Duc; DeBose-Boyd, Russell A.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.Item Regulation of Hepatic Cholesterol Homeostasis Through Accelerated Degradation of HMG CoA Reductase(2017-04-06) Hwang, Seonghwan; Liang, Guosheng; DeBose-Boyd, Russell A.; Bruick, Richard K.; Scherer, PhilippCholesterol biosynthesis is rigorously controlled by negative feedback regulation. This reaction occurs, in part, through sterol-accelerated degradation of HMG CoA reductase (HMGCR), which catalyzes the rate-limiting step in cholesterol biosynthesis. The molecular mechanisms for the degradation of HMGCR have been actively investigated; however, the physiological relevance of the degradative regulation in animals is unclear. The current study investigates the role of sterol-accelerated degradation of HMGCR in overall regulation of HMGCR protein and cholesterol homeostasis in the liver. This was achieved by utilizing two mouse models: (1) liver-specific transgenic mice expressing the membrane domain of HMGCR, which is necessary and sufficient for sterol-regulated degradation of HMGCR in cultured cells and (2) knock-in mice expressing mutant HMGCR that is resistant to sterol-induced ubiquitination. These models were subjected to various feeding regimens known to modulate Insig and Scap, key players in feedback regulation of HMGCR. Cholesterol replenishment accelerates degradation of HMGCR in the liver of transgenic animals, whereas deprivation of sterols by lovastatin administration suppresses degradation of HMGCR. Ubiquitination-resistant HMGCR accumulated in the liver and resulted in the elevation of hepatic cholesterol, indicating degradation plays a significant role in the in vivo regulation of the enzyme and cholesterol homeostasis. This study further explored the physiological settings other than changing cholesterol status that may modulate the degradation of HMGCR in the two mouse models. As cholesterol synthesis is an oxygen-consumptive process, I determined the link between oxygen sensing and feedback control of cholesterol synthesis. In cultured human fibroblasts, stabilization of oxygen-sensitive transcription factor, hypoxia-inducible factor-1α (HIF-1α) directly activates transcription of INSIG-2 gene. Insig-2 inhibits cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent degradation of HMGCR. Hepatic levels of Insig-2 mRNA are enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in liver stimulates HMGCR degradation through a reaction that requires the protein's prior ubiquitination and the presence of Insig-2. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events in the cellular adaptation to hypoxic stress. Overall, the current study provides evidence supporting the physiological significance of the accelerated degradation of HMGCR in cholesterol homeostasis.