Sterol Carrier Protein-2 Overexpression Enhances Sterol Cycling and Inhibits Cholesterol Ester Synthesis and High Density Lipoprotein Cholesterol Secretion

Abstract

Recent data indicate that sterol carrier protein-2 (SCP-2) functions in the rapid movement of newly synthesized cholesterol to the plasma membrane (Puglielli, L., Rigotti, A., Greco, A. V., Santos, M. J., and Nervi, F. (1995) J. Biol. Chem. 270, 18723-18726). In order to further characterize the cellular function of SCP-2, we transfected McA-RH7777 rat hepatoma cells with a pre-SCP-2 cDNA expression construct. In stable transfectants, pre-SCP-2 processing resulted in an 8-fold increase in peroxisomal levels of SCP-2. SCP-2 overexpression increased the rates of newly synthesized cholesterol transfer to the plasma membrane and plasma membrane cholesterol internalization by 4-fold. There was no effect of SCP-2 overexpression on the microsomal levels of acyl-CoA:cholesterol acyltransferase and neutral cholesterol ester (CE) hydrolase; however, in the intact cell, CE synthesis and mass were reduced by 50%. SCP-2 overexpression also reduced high density lipoprotein-cholesterol secretion and apoA-I gene expression by 70% and doubled the rate of plasma membrane desmosterol conversion to cholesterol. We conclude that SCP-2 overexpression enhances the rate of cholesterol cycling, which reduces the availability of cholesterol for CE synthesis and alters the activity of a cellular cholesterol pool involved in regulating apoA-Imediated high density lipoprotein cholesterol secretion. The net result of these changes in cholesterol metabolism is a 46% increase in plasma membrane cholesterol content, the implications of which are discussed. Cellular free cholesterol is predominantly located in the plasma membrane (reviewed in Ref. 1). Cellular cholesterol content, however, is determined by the concerted action of intracellular enzymes and regulatory proteins as follows: ACAT 1 which catalyzes the synthesis of CE, sterol regulatory element binding proteins, which regulate the transcription of a number of genes involved in cholesterol metabolism (2, 3), and various cell type-specific metabolic reactions, e.g. lipoprotein secretion, steroidogenesis, and bile acid synthesis. Since cholesterol is highly insoluble in an aqueous environment, it has been postulated that sterol carrier protein-2 (SCP-2) regulates the movement and thus the availability of cholesterol for different cellular processes (4 -6). The evidence supporting this contention was initially derived in large part from studies demonstrating that the addition of purified SCP-2 stimulated the in vitro conversion of sterol intermediates to cholesterol (7) and cholesterol to 7␣-hydroxycholesterol (8) steroid hormones (9 -13) and cholesterol ester (14). More recent studies indicate that SCP-2 gene expression is regulated by changes in cellular cholesterol content (15-17); however, in these studies, a direct role in cholesterol trafficking and esterification was not demonstrated. The strongest support for a role in cellular cholesterol metabolism comes from studies on the role of SCP-2 in steroidogenesis. SCP-2 gene expression is coordinately regulated with steroid hormone syn...

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

“…We hypothesize that the triggering event involved in the multiple effects of hepatic SCP-2 overexpression on lipid metabolism in mice was the increased FA and cholesterol cycling and intracellular cholesterol and phosphatidylcholine redistribution. This possibility is consistent with the results of previous studies in SCP-2-transfected cultured rat hepatoma cells (23) and in the whole animal (26). It was apparent that the velocity of the bidirectional flux of cholesterol and phosphatidylcholine between endoplasmic reticulum and plasma membranes was critically dependent upon SCP-2 expression levels.…”

“…Experiments in cultured cell systems transfected with cDNAs encoding for the SCP-2/sterol carrier protein X (SCP-X) products demonstrated the role of these proteins in microsomal membrane utilization of FA for phospholipids, triglyceride, and cholesterol ester synthesis (19)(20)(21)(22). Overexpression of SCP-2 in rat hepatoma cells enhanced intracellular cholesterol cycling, increased plasma membrane cholesterol content, and inhibited cholesterol esterification and HDL production (23). In addition, studies in mice with disruption of the SCP-2/SCP-X gene have shown a failure in the oxidation of 2-methyl-branched FA and the side chain of cholesterol.…”

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…The physiological impact of cholesterol binding proteins such as SCP-2 and L-FABP on cholesterol uptake, esterification, and excretion into bile was confirmed by studies with SCP-2 transgenic (i.e. overexpressing or antisense cDNA treated) mice and rats (177-179) Studies with radiolabeled cholesterol were consistent with many of the findings shown with the fluorescent DHE and other sterols in cultured cells (43,45,172,(190)(191)(192)(193)(194)(195)(196)(197). In one study, it was shown that overexpression of L-FABP in L-cell fibroblasts enhanced the transfer or radiolabled cholesterol from the plasma membrane to the endoplasmic reticulum for esterification (194).…”

ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

“…of Medicine, Clinical Nutrition Research Unit, Section of Gastroenterology, Univ. of Chicago, Chicago, IL), were maintained as described (27). For indirect immunofluorescence (see below), cells were grown on glass cover slip chambers and prepared as described earlier (28).…”