Cholesterol and 25-Hydroxycholesterol Inhibit Activation of SREBPs by Different Mechanisms, Both Involving SCAP and Insigs

Abstract

The current paper demonstrates that cholesterol and its hydroxylated derivative, 25-hydroxycholesterol (25-HC), inhibit cholesterol synthesis by two different mechanisms, both involving the proteins that control sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate genes encoding enzymes of lipid synthesis. Using methyl-␤-cyclodextrin as a delivery vehicle, we show that cholesterol enters cultured Chinese hamster ovary cells and elicits a conformational change in SREBP cleavage-activating protein (SCAP), as revealed by the appearance of a new fragment in tryptic digests. This change causes SCAP to bind to Insigs, which are endoplasmic reticulum retention proteins that abrogate movement of the SCAP⅐SREBP complex to the Golgi apparatus where SREBPs are normally processed to their active forms. Direct binding of cholesterol to SCAP in intact cells was demonstrated by showing that a photoactivated derivative of cholesterol cross-links to the membrane domain of SCAP. The inhibitory actions of cholesterol do not require the isooctyl side chain or the ⌬5-double bond of cholesterol, but they do require the 3␤-hydroxyl group. 25-HC is more potent than cholesterol in eliciting SCAP binding to Insigs, but 25-HC does not cause a detectable conformational change in SCAP. Moreover, a photoactivated derivative of 25-HC does not cross-link to SCAP. These data imply that cholesterol interacts with SCAP directly by inducing it to bind to Insigs, whereas 25-HC works indirectly through a putative 25-HC sensor protein that elicits SCAP-Insig binding.Nearly 30 years ago, during early studies of feedback inhibition of cholesterol synthesis in cultured cells, it was noted that oxygenated sterols such as 25-hydroxycholesterol were more than 50-fold more potent than cholesterol in reducing the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-controlling enzyme in cholesterol biosynthesis (1-4). These experiments were conducted by dissolving sterols in ethanol and adding them to protein-containing aqueous culture media in which cholesterol forms an amorphous suspension and thus has poor access to the interior of the cell. When cholesterol was delivered to cells in low density lipoprotein (LDL), 1 a physiologic carrier that enters cells through LDL receptors, the ability of cholesterol to suppress 3-hydroxy-3-methylglutaryl-CoA reductase was enhanced (5). Later, when methods were devised to reconstitute LDL with sterol esters, it was observed that 25-hydroxycholesterol was only about 5-fold more potent than cholesterol when both sterol esters were reconstituted into LDL and delivered through LDL receptors (6). The question of whether cholesterol itself is a regulator or whether it must be converted to an oxygenated metabolite, like 25-hydroxycholesterol, remained unresolved (7). In view of this ambiguity, studies of feedback regulation in our laboratory have generally used a mixture of cholesterol and 25-hydroxycholesterol in a 10:1 molar ratio added in ethanol.In recent years...

Regulation of Human Cytochrome P450 4F2 Expression by Sterol Regulatory Element-binding Protein and Lovastatin

“…This sterol derivative blocks transfer of the membrane-bound SREBP-2 precursor from endoplasmic reticulum to Golgi, and prevents proteolytic cleavage that produces the active nuclear form by causing SCAP to bind to Insig (42). Although no statistically significant affect on endogenous CYP4F2 mRNA expression was seen when the HepG2 cells were treated with 25-OH alone, lovastatin induction of endogenous CYP4F2 gene expression was completely suppressed by the co-administration of 25-OH (Fig.…”

Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol

“…Previous studies have demonstrated that the mechanism for blocking SREBP processing by 25-OH is indirect. 25-OH binds to a putative protein that enhances SCAP-INSIG binding and thus promotes retention of pSREBP within the endoplasmic reticulum (32). It is possible that inhibition of pSREBP-1 processing by OSCi-induced 24(S),25-epoxy acts through a similar mechanism.…”

“…The structure of TO-901317 (37) differs substantially from oxysterols and cholesterol (32), which potentially does not allow TO-901317 to bind SCAP or the putative SCAP-INSIG binding protein. The essential part of the sterol that mediates binding to SCAP or a SCAP-INSIG-binding protein was shown to be the 3␤-hydroxyl group (32), which is absent from TO-901317 (37).…”

“…Previously, 25-OH has been shown to decrease mature nSREBP-1 protein levels in Chinese hamster ovary cells (32) and CaCo-2 intestinal epithelial cells (33). Therefore, we sought to confirm this finding in THP-1 macrophages.…”

Hypercholesterolemia in rats with hepatomas: Increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol

“…35,36 Under normal conditions, high concentrations of cholesterol in tissue stimulate oxysterol production, and the oxysterols, as well as cholesterol, downregulate cholesterol biosynthesis 37 by inhibiting HMGCR, the rate-limiting enzyme in the cholesterol biosynthetic pathway. In McA-RH7777 hepatomas, however, HMGCR was markedly upregulated despite the increased concentrations of both oxysterols and cholesterol.…”