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Journal of Lipid Research, Vol. 50, 1881-1888, September 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol

Yvonne Lange^1,*, Theodore L. Steck, Jin Ye^*, Michael H. Lanier, Vasumathi Molugu and Daniel Ory

^* Department of Pathology, Rush University Medical Center, Chicago, IL 60612
Department of Biochemistry and Molecular Biology, University of Chicago, 920 E. 58th Street, Chicago, IL 60637
Departments of Medicine, Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110

¹ To whom correspondence should be addressed: e-mail: ylange{at}rush.edu

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins. Here, we investigated how plasma membrane (PM) cholesterol might regulate 27-hydroxycholesterol (HC) biosynthesis in cultured fibroblasts. We showed that PM cholesterol was a major substrate for 27-HC production. Biosynthesis commenced within minutes of loading depleted cells with cholesterol, concurrent with the rapid inactivation of hydroxy-3-methylglutaryl CoA reductase (HMGR). 27-HC production rose 30-fold in normal and Niemann-Pick C1 fibroblasts when PM cholesterol was increased by 60%. 27-HC production was also stimulated by 1-octanol, which displaces PM cholesterol from its phospholipid complexes and thereby increases its activity (escape tendency) and elevates its intracellular abundance. Conversely, lysophosphatidylserine and U18666A inhibited 27-HC biosynthesis and the inactivation of HMGR, presumably by reducing the activity of PM cholesterol and, therefore, its circulation to mitochondria. We conclude that, in this in vitro system, excess (active) PM cholesterol rapidly reaches mitochondria where, as the rate-limiting substrate, it stimulates 27-HC biosynthesis. The oxysterol product then promotes the rapid degradation of HMGR, along with other homeostatic effects. The regulation of 27-HC production by the active excess of PM cholesterol can thus provide a feedback mechanism in the homeostasis of PM cholesterol.

Supplementary key words homeostasis • endoplasmic reticulum • oxysterol • feedback • Niemann-Pick

Abbreviations: ER, endoplasmic reticulum; HC, hydroxycholesterol; HMGR, hydroxy-3-methylglutaryl CoA reductase; HPCD, 2-β-hydroxypropylcyclodextrin; LPS, 1-palmitoyl-lysophosphatidylserine; medium A, DMEM plus 5% lipoprotein-deficient serum; NPC1, Niemann-Pick C type 1; PBS, 0.15 M NaCl + 5 mM NaP_i, pH 7.4; PM, plasma membrane; SREBP, sterol regulatory element-binding protein

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