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-I-mediated 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.
Highlights
Cellular free cholesterol is predominantly located in the plasma membrane
In order to determine if the effect of sterol carrier protein-2 (SCP-2) on cholesterol ester (CE) synthesis could be reversed by treatment with oxysterol, we examined the effect of 25-hydroxycholesterol on CE synthesis in SCPhigh and NEO cells
In SCPhigh cells the rate of cholesterol internalization paralleled the rate of cholesterol transfer to the plasma membrane, suggesting that the latter event determines the rate of bi-directional cholesterol flux
Summary
Materials—[3H]Acetic acid (sodium salt, 5.3 Ci/mmol), [14C]cholesterol (54 mCi/mmol), [3H]cholesterol (51 Ci/mmol), [3H]cholesteryl oleate (66 Ci/mmol), [3H]oleic acid (14 Ci/mmol), [14C]oleoyl coenzyme A (53 mCi/mmol), and 35S-Protein Labeling Mix (43.5 TBq/mmol) were purchased from DuPont NEN. [3H]Desmosterol was prepared by incubating McA-RH7777 cells with [3H]acetate as described previously [36]. [3H]Desmosterol was prepared by incubating McA-RH7777 cells with [3H]acetate as described previously [36]. Oxidized sterol standards were prepared by treatment with cholesterol oxidase as described previously [37]. Twenty-four to forty-eight hours prior to the initiation of experiments, medium was removed and fresh medium containing either 10% FBS or 5% (v/v) lipoprotein-deficient fetal bovine serum, prepared as described [42], was added. Samples (100 g) were separated by 12.5% SDS-PAGE and electroblotted to polyvinylidene difluoride membranes (Immobilon, Millipore) as described previously [41]. A polyclonal rabbit anti-rat SCP-2 antiserum was incubated with membranes as described previously and detected with an anti-rabbit horseradish peroxidase-labeled secondary antibody-catalyzed chemiluminescence reaction (Amersham Corp.) [41]. Sections with thickness in the range of 70 –100 nm were collected on copper grids and stained with anti-SCP-2 antisera
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