Sterol Carrier Protein-2 Alters High Density Lipoprotein-mediated Cholesterol Efflux

Abstract

were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.Although the HDL-mediated 1 steps of cholesterol transfer from the cell surface membrane and subsequent fate of cholesterol in the vasculature have been extensively studied, much less is known about intracellular components of cholesterol efflux (reviewed in Refs. 1-5). Plasma membrane cholesterol is distributed into multiple pools or domains (reviewed in Refs. 6 and 7). It is now recognized that there may be a connection between such domains and HDL receptor-mediated reverse cholesterol transport (reviewed in Refs. 7-9). The transbilayer distribution of cholesterol in plasma membranes is asymmetric, with the cholesterol enriched 400% in the cytofacial leaflet versus exofacial leaflet (reviewed in Refs. 10 -14). Transbilayer movement of cholesterol across the plasma membrane appears fast (t1 ⁄2 ϭ 1-6 min; reviewed in Ref. 10). Plasma membrane cholesterol is also distributed into lateral cholesterol-rich and -poor membrane domains (reviewed in Refs. 6 and 10). Most of the cholesterol in the plasma membrane is localized in lateral domains that are, for the most part, relatively inert in terms of transfer kinetics (i.e. t1 ⁄2 ϭ hours to days), and movement between such domains is also slow. However, a small pool of plasma membrane cholesterol appears highly dynamic (reviewed in Ref. 6) and is associated with cholesterol-rich, HDL receptor containing microdomains called caveolae (reviewed in Refs. 8, 9, 15, and 16). Molecular details of cholesterol entry/ exit, cholesterol organization, and mechanism(s) of cholesterol transbilayer movement in caveolae remain to be determined. Likewise, the relationships between caveolae, "rafts," and other cholesterol-rich plasma membrane microdomains is not yet clear (reviewed in Ref. 9).The intracellular steps preceding cellular cholesterol efflux include transfer of cholesterol from the Golgi, endoplasmic reticulum, and lipid droplets to the plasma membrane (reviewed in Refs. 1, 9, and 17). The time frame of bidirectional vesicular transfer of cholesterol between plasma membranes and Golgi has a t1 ⁄2 of 10 -20 min (reviewed in Refs. 8,18,and 19). Alternately, molecular cholesterol transfer, mediated by cholesterol-binding proteins in the cytoplasm, occurs much faster (t1 ⁄2 near 1-2 min) from the lysosome (exogenous cholesterol) ...

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…The significance of the above findings in living cells was confirmed with DHE and other fluorescent sterols in a variety of cultured cells including L-cells, CHO cells, macrophages, and hepatocyte derived cell lines (39,43,45,160,(174)(175)(176). Studies with transfected L-cells showed that uptake and efflux of fluorescent sterols was highly dependent on the expression of cholesterol binding proteins (e.g.…”

“…SCP-2, ADRP) or their real-time uptake, distribution, and efflux dynamics in living cells. One exception is 22-NBD-cholesterol which has been shown to be not only bound with high affinity (nM K d s) by SCP-2 and ADRP, but with orientation within the binding site similar to cholesterol-suggesting that this probe may be suitable for study of intracellular cholesterol trafficking (4,(39)(40)(41)(42)(43)(44). Indeed, 22-NBDcholesterol has proven useful for real-time imaging of HDL-mediated cholesterol uptake, efflux, and intracellular trafficking in living cells such as L-cell fibroblasts as well as rat macrophages and lymphocytes (43,45,46).…”

“…Sterol carrier protein-2 (SCP-2) overexpression facilitated (i.e. decreased t 1/2 of efflux) of protein mediated NBD-cholesterol cytosolic efflux (43). Concomitantly SCP-2 overexpression decreased the slower, vesicular cholesterol transfer -likely by binding and sequestering ligands (i.e.…”

“…Concomitantly SCP-2 overexpression decreased the slower, vesicular cholesterol transfer -likely by binding and sequestering ligands (i.e. phosphatidylinositides, fatty acyl CoAs) that regulate vesicular trafficking (43,(47)(48)(49)(50)(51)(52)(53).…”

“…Instead, DHE from commercial (Sigma, St. Louis, MO) as well as laboratory sources is chemically synthesized by an adaptation of a 50 year old method (64,(69)(70)(71). However, synthetic DHE prepared by this established method, especially that obtained from commercial sources, may contain a significant sterol impurity, varying from batch to batch, and as high as 20-40% (43,70,71). It is important to note that not only the quantity, but also the chemical structure of the sterol and the presence of cholesterol oxidation products are known to significantly affect the structure and/or function of model membranes (rev.…”