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

Abstract

This study addresses the regulation of the biosynthesis of oxysterol signal molecules. Side chain oxysterols such as 24-, 25-, and 27-hydroxycholesterol (HC) are derived from cholesterol in the endoplasmic reticulum (ER) and mitochondria and trigger downregulation of the abundance of cell cholesterol through diverse pathways ( 1-4 ). In particular, the association of oxysterols with nuclear liver X receptors (LXR) promotes the expression of ATP-binding cassette (ABC) transport proteins that transfer excess cholesterol out of cells such as macrophages ( 5 ). Furthermore, because the oxysterol derivatives are more water-soluble than cholesterol, they readily exit cells and are transported via circulating lipoproteins to the liver for conversion to bile acids and excretion ( 6, 7 ). In addition, side-chain oxysterols promote the interaction of Insig proteins with sterol regulatory element-binding protein (SREBP) cleavage-activating protein-SREBP complexes; this action sequesters SREBP in the ER and, as a result, reduces the expression of genes for cholesterol accretion ( 8 ). By activating Insig, side-chain oxysterols also stimulate the proteolysis of hydroxy-3-methylglutaryl CoA reductase (HMGR) through a ubiquitination-proteasomal pathway ( 9, 10 ).The mechanism by which oxysterol biosynthesis is set according to the level of cell cholesterol so as to execute these homeostatic functions is obscure. Indeed, despite the cogency of the oxysterol hypothesis, the physiological relevance of the underlying in vitro fi ndings has long been debated ( 1, 11 ). To carry out its postulated role in vivo, oxysterol production should represent the level of cellular 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 fi broblasts. 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 fi broblasts 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 ( Press, April 28, 2009 DOI 10.1194 Regulation of fi broblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol Abbreviations: ER, endoplasmic reticulum; HC, hydroxycholesterol; HMGR, hydroxy-3-methylglutaryl CoA reductase; HPCD, 2-␤ -...

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

“…ABCA3 has previously been shown to be involved in early steps of late endosomal biogenesis ( 15 ), and both MLN64 and ABCA3 have higher expression in lung where ABCA3 has been shown to be important for the secretion of late endosomal lipids at the PM. Cholesterol destined for the mitochondria passes through the PM ( 36 ). MLN64 is involved in the egress of cholesterol from LE to mitochondria ( 4 ) in a process that is independent of NPC1 ( 7 ), indicating that MLN64 may be involved in the traffi cking of cholesterol upstream of NPC1-and ORP1L-containing late endosomes as an additional step for LE cholesterol export.…”

Cholesterol through the Looking Glass

“…5A). We employed a concentration of U18666A (4.5 M) that has previously been shown to inhibit 27-hydroxycholesterol formation in the mitochondria and to increase HMGCR activity in the ER (38). It is also noteworthy that reducing cell cholesterol further by cyclodextrin treatment did not increase SREBP-2 target gene expression (as was also seen in the CD control in Fig.…”

“…Thus, Lange et al (38) have shown that following cholesterol enrichment with a deuterated cholesterol probe, the endogenous cholesterol is internalized prior to the exogenously loaded cholesterol complexed to cyclodextrin. Therefore, it might be argued that ACAT would first have been presented with endogenous cholesterol and raises the question of why this esterification was not detected with ent-cholesterol in Fig.…”

“…By contrast, no such band was observed under our basal cholesterolstarved condition (LPDS with statin), even when the phosphorimage was exposed for five times as long (2 weeks versus 3 days). To address this issue further, we took a lead from the work of Lange et al (30,38) (31,40). Under these conditions, very little of the pulsed [ 3 H]cholesterol is esterified (Fig.…”

Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

“…a permanently activated Er may well be the consequence of an association with endogenously produced SErms, such as 27Ohc. through the metabolism of profusely available plasma membrane cholesterol, many cells produce and secrete 27Ohc in their neighborhood, as demonstrated in the case of fibroblasts (28). The infiltration of macrophages is an indicator of poor prognosis in patients with a mammary gland affected by a tumor (29).…”