Abstract
The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.
Highlights
From the $Divisionof Pharmacology and Medical Chemistry, Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, Cincinnati, Ohio45267-0004 and the )IDepartment of Biochemistry, University of New Mexico
L-cells would provide a good model for transfection with cDNA encoding L-FABP. These (L-FABP)
Rat liver recombinant L-FABP, endogenous mouse fibroblast FABP, and rat liver SCP-2 were purified as described under "Experimental Procedures." It was not necessary topurify an endogenous SCP-2 since anti-rat liver SCP2 IgG cross-reacted with the endogenous SCP-2, but notwith the other proteins.The purified proteins were used to produce rabbit IgG antibodies specific for the respective antigens
Summary
Endogenous L-cell LipidTransfer Proteins-As pointed out in the Introduction, all mammalian cells far tested appear to contain intracellular lipid transfer proteins. The specific antibodies and standardantigens were used in Western blot and enzyme-linked immunosorbant assays to determine the levels of L-FABP, endogenous FABP, and SCP-2in cytosols from control and transfected L-cells. Three lines of evidence indicate that sterol uptake was significantly enhanced in L-cells expressing 0.34% of cytosolic protein as L-FABP. 3) HPLC of lipid extracts taken from cells cultured with serum containing medium and 10 pg of dehydroergosterol/ml confirmed that the increase in fluorescence noted above was due to increased dehydroergosterol uptake in the transfected cells expressing 0.34% of cell cytosol as L-FABP. Lipid Composition of Plasma Membranes from L-cells Expressing L-FABP-Because the above dataareconsistent extracted from washed cells, resolved by high performance thin layer chromatography (radiolabel) oHr PLC (dehydroergosterol), and quantitated (scintillationcounting or quantitativeHPLC with ergosterol internal standard) to determine sterol uptake. The data on fluorescent sterol uptakeby whole cells and on the sterol content ancdomposition of isolated plasma nmol/mg protein
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