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

Yvonne Lange,Theodore L Steck,Jin Ye,Michael H Lanier,Vasumathi Molugu,Daniel Ory

doi:10.1194/jlr.m900116-jlr200

Abstract

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 approximately 30-fold in normal and Niemann-Pick C1 fibroblasts when PM cholesterol was increased by approximately 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.

Highlights

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins
LDL cholesterol is impeded in Niemann-Pick C type 1 (NPC1)-deficient cells, the movement of plasma membrane (PM) cholesterol to the endoplasmic reticulum (ER), as promoted by cholesterol loading via cyclodextrin or by 25-HC displacement, is functionally normal in the mutant cells [14, 23]. These findings suggest that the movement of PM cholesterol to ER and mitochondrial membranes is not limited by NPC1-mediated pathways; rather, the phenotype in NPC1 cells is related to impairment of the exit of endolysosomal cholesterol
Cholesterol biosynthesis is inactivated just as rapidly following a small jump in PM cholesterol; this occurs in the ER through the proteolytic degradation of the rate limiting enzyme, hydroxy-3-methylglutaryl CoA reductase (HMGR) [see Fig. 3B and [17]]

Summary

Introduction

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins. Elevating cell cholesterol using HPCD-cholesterol complexes increased 27-HC production ‫ف‬6-fold, to as high as 13 ng/mg protein/h, with no sign of reaching a maximum.

Results

Conclusion