INHIBITION OF CELLULAR CHOLESTEROL EFFLUX BY 25-HYDROXYCHOLESTEROL

The effect of oxysterols on efflux of cholesterol from mouse L-cell fi broblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hy droxycholesterol (25-OHC) either during labeling of the cells with [H- 3]cholesterol or during equilibration after labeling. Subsequently, th e release of [H-3]cholesterol into medium containing 0.2 mg HDL(3)/ml was measured and the fractional release of cellular [H-3]cholesterol w as calculated. Pretreatment with 25-OHC (1 mu g/ml) caused a 30% reduc tion in [H-3]cholesterol efflux from L-cells during 8 h of incubation with HDL(3). 25-OHC also inhibited cholesterol efflux from Fu5AH and J 774 cells, but the effect was less marked. There was only a small, non significant reduction of efflux from EA.hy 926 cells. The mechanism of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with si gnificant effects seen at 25-OHC concentrations as low as 50 ng/ml. Th e half-time for cholesterol efflux from 25-OHC-treated cells (5 mu g/m l) was 13.0 +/-: 3.3 h compared to 5.7 +/- 1.0 in control cells, corre sponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxych olesterol, and 22(S)-hydroxycholesterol also inhibited [H-3]cholestero l efflux from L-cells significantly, but to a lesser degree. 25-Hydrox ycholesterol (5 mu g/ml) reduced efflux from both normal and cholester ol-enriched cells by 31 and 14%, respectively. Inhibition of efflux wa s similar when reconstituted HDL(3)-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidyl choline vesicles were use d as cholesterol accepters instead of HDL. The content of phospholipid s, cholesterol and the FC/PL ratio of intact cells and from isolated p lasma membrane vesicles were the same for control and 25-OHC-treated c ells. Efflux of [H-3]cholesterol from plasma membranes isolated from 2 5-OHC-treated cells was 20% less than efflux from membranes from contr ol cells. The difference in efflux observed in intact cells is partial ly explained by the reduction in efflux from the plasma membrane. In c onclusion, our studies suggest that oxysterols, especially 25-hydroxyc holesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse choles terol transport.